BE1022345A9 - USPA2 PROTEIN CONSTRUCTS AND USES THEREOF - Google Patents

Abstract

The present invention provides compositions comprising the ubiquitous surface protein A2 (UspA2) of Moraxella catarrhalis (M. catarrhalis). More particularly, the present application relates to UspA2 protein constructs and immunogenic compositions comprising constructs, vaccines comprising such immunogenic compositions and their therapeutic uses. The invention further provides compositions comprising UspA2 in combination with at least one Haemophilus influenzae antigen, immunogenic compositions comprising the antigens, vaccines comprising such immunogenic compositions, and their therapeutic uses.

Description

USPA2 PROTEIN CONSTRUCTS AND THEIR USES Field of the invention

The present invention provides compositions comprising the ubiquitous surface protein A2 (UspA2) of Moraxella catarrhalis (M. catarrhalis, M. cat.). More particularly, the present invention relates to UspA2 protein constructs and immunogenic compositions comprising constructs, vaccines comprising such immunogenic compositions and their therapeutic uses.

Background of the invention

Ubiquitous surface protein A2 (UspA2) is a trimeric autotransporter which appears as a licker remanufacturer on electron micrographs (Hoiczyk et al., EMBO J. 19: 5989-5999 (2000)). It is composed of an N-terminal head followed by a stem ending in an amphipathic helix and a C-terminal membrane domain. (Hoiczyk et al., EMBO J. 19: 5989-5999 (2000)). UspA2 contains a very well conserved domain (Aebi et al., Infection & Immunity 65 (11) 4367-4377 (1997)), which is recognized by a monoclonal antibody whose protective effect has been demonstrated after passive transfer into a model. test method with Moraxella catarrhalis in mice (Helminnen et al., J Infect Dis., 170 (4): 867-72 (1994)).

UspA2 has been shown to interact with host structures and extracellular matrix proteins such as fibronectin (Tan et al., J Infect Dis. 192 (6): 1029-38 (2005)) and laminin ( Tan et al., J Infect Dis., 194 (4): 493-7 (2006)), suggesting that it may play a role at an early stage of Moraxella catarrhalis infection.

UspA2 also appears to be involved in the ability of Moraxella catarrhalis to resist the bactericidal activity of normal human serum. (Attia AS et al Infect Immun 73 (4): 2400-2410 (2005)). It (i) binds the C4bp complement inhibitor, allowing Moraxella catarrhalls to inhibit the classical complement system, (ii) prevents activation of the alternative complement pathway by absorbing C3 from the serum, and (iii) interferes with with the terminal stages of the complement system, the membrane attack complex (MAC), by binding the complement-regulating vitamin C protein, (de Vries et al., Microbiol Mol Biol Rev. 73 (3): 389-406 (2009)). )).

Moraxella catarrhalis is an important and common respiratory pathogen that has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease (COPD) in adults. (Sateesh et al., Journal of Chronic Obstructive Pulmonary Disease 3: 109-115 (2006)).

There is a need for vaccines against Moraxella catarrhalis.

Brief summary of the invention

As a first aspect, the present invention provides the proteins of formula (I).

wherein: A represents UspA2 of Moraxella catarrhalis or an immunogenic fragment thereof;

Ri represents an amino acid; m is 0, 1 or 2; B represents a histidine; and n is 0, 1, 2, 3, 4, 5 or 6.

As a second aspect, the present invention provides immunogenic compositions comprising proteins of formula (I) and proteins of the invention. The composition may include

in addition a pharmaceutically acceptable adjuvant. The composition may comprise an excipient.

In a third aspect, the present invention provides a method of treating or preventing an ailment or disease caused in whole or in part by Moraxella catarrhalis. The method comprises administering to a subject in need thereof a therapeutically effective amount of a protein of formula (I) or a protein of the invention.

In a fourth aspect, the present invention provides a method of treating or preventing otitis media. The method comprises administering to a subject in need thereof a therapeutically effective amount of a protein of formula (I) or a protein of the invention.

In a fifth aspect, the present invention provides a method of treating or preventing exacerbations in obstructive chronic bronchopneumopathies. The method comprises administering to a subject in need thereof a therapeutically effective amount of a protein of formula (I) or a protein of the invention.

In a sixth aspect, the present invention provides a method of treating or preventing pneumonia. The method comprises administering to a subject in need thereof a therapeutically effective amount of a protein of formula (I) or a protein of the invention.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising a protein of formula (I) or a protein of the invention for use in the treatment or prevention of an ailment or disease caused in whole or in part by Moraxella catarrhalis. The pharmaceutical compositions may further comprise a pharmaceutically acceptable adjuvant.

In an eighth aspect, the present invention provides nucleic acids encoding the proteins of the invention.

In a ninth aspect, the present invention provides a method for producing nucleic acids of the invention.

In a tenth aspect, the present invention provides a composition comprising at least one antigen from Moraxella catarrhalis and at least one antigen from Haemophilus influenzae. The composition may further comprise a pharmaceutically acceptable adjuvant. The composition may comprise an excipient.

In a further aspect, the present invention provides a method of treating or preventing an ailment or disease caused in whole or in part by Moraxella catarrhalis and / or Haemophilus influenzae. The method comprises administering to a subject in need thereof a therapeutically effective amount of a protein of formula (I) or a protein of the invention.

In another aspect, the present invention provides a method of treating or preventing exacerbations in obstructive chronic bronchopneumopathies. The method comprises administering to a subject in need thereof a therapeutically effective amount of a protein of formula (I) or a protein of the invention and a therapeutically effective amount of at least one antigen. from Haemophilus influenzae.

The present invention also provides a pharmaceutical composition comprising a protein of formula (I) or a protein of the invention for use in the treatment or prevention of an ailment or disease caused in whole or in part by Moraxella catarrhalis in combination with at least one antigen from Haemophilus influenzae. The pharmaceutical compositions may further comprise a pharmaceutically acceptable adjuvant. Other aspects of the present invention are described in the detailed description of particular embodiments, examples, and claims that follow.

Brief description of the drawings

Figure 1 - Typical fermentation profile with high-density cell induction (HCDI) methods and parameters monitored in 20 l fermenter with a fed-batch.

FIG. 2 - Typical fermentation profile with low density cell induction (LCDI) methods and parameters monitored in a 20 l fermenter with a fed-batch.

Figure 3 - UspA2 yield from protein constructs MC-001, MC-002, MC-004, MC-005, MC-006, MC-007, MC-008 and MC-010 evaluated in a fermenter; data from Table 4.

Figure 4 - Molecular weight distribution of purified MC-005 determined by sedimentation rate by analytical ultracentrifugation. The majority of the protein is in the form of a trimer, with a small proportion of higher molecular weight oligomers which may be a dimer of trimers. PM = molecular weight. kDa = kilodalton.

Figure 5 - Molecular weight distribution of purified MC-001 determined by the rate of sedimentation by analytical ultracentrifugation. The majority of the protein is in the form of a trimer.

Figure 6 - Molecular weight distribution of purified MC-001 determined by sedimentation rate by analytical ultracentrifugation. The sample has multiple species and is highly polydispersed. The sedimentation coefficient of the main species detected does not correspond to one of the trimers normally detected in the other batches.

Figure 7 - Molecular weight distribution of purified MC-001 determined by the rate of sedimentation by analytical ultracentrifugation. The majority of the protein is in the form of a trimer.

Figure 8 - Molecular weight distribution of purified MC-007 determined by sedimentation rate by analytical ultracentrifugation. The majority of the protein is in the form of a trimer.

Figure 9 - Spectra of circular dichroism (CD) in the far UV of the UspA2 constructs giving an indication of the secondary structures of the proteins.

Figure 10 - Follow-up of secondary structures by circular dichroism (CD) during thermal unfolding of MC-005 (UspA2Ahelice + 6His). Visual analysis of the spectra clearly shows that the protein loses most of its secondary structures at 33 ° C.

Figure 11 - Follow-up of secondary structures by circular dichroism (CD) during thermal unfolding of MC-007 (UspA2 full helix + 6His). The visual analysis of the spectra shows that the loss of secondary structure is slower compared to the construction without propeller. Structural changes are detectable after heating at 33 ° C, but complete unfolding appears to occur between 35 ° C and 37 ° C.

Figure 12 - MALDI Spectrum of MC-001 lot opt-01. The mass observed at 57427 Da may be consistent with the demethionylated protein, while the peak at 57620 Da could correspond to the complete protein.

Figure 13 - MALDI spectrum of MC-011 lot BMP37. The observed mass may be consistent with the demethionylated protein. The other two peaks at +186 Da and +366 Da are not identified.

Figure 14 - Protective efficacy of MC-001 and MC-007 in a mouse model of colonization of the lung.

Figure 15 - UspA2-directed antibody response induced after intramuscular administration in mice, where PII and PI11 indicate, respectively, anti-IgG levels in the sera taken at day 28 (after II) and at day 42 (after III ).

Figure 16 - UspA2-induced bactericidal titres against a homologous strain formulated with different adjuvants (AS01E, AS04C and AIPO4).

Figure 17 - UspA2-directed antibody response induced after intramuscular administration in mice, using different formulations of antigens and adjuvants.

Figure 18 - UspA2-induced bactericidal titres against a homologous strain, using different formulations of antigens and adjuvants.

Figure 19 - Induced IgG response against PD in mice by the PD-PEPilA-UspA2 vaccine (a trivalent NTHi-Ms cat vaccine), formulated with different adjuvants.

Figure 20 - IgG response induced against PE in mice by the PD-PEPilA-UspA2 vaccine (a trivalent vaccine NTHi-MS cat.), Formulated with different adjuvants.

Figure 21 - PilA-induced IgG response in mice by the PD-PEPilA-UspA2 vaccine (a trivalent NTHi-Ms cat vaccine), formulated with different adjuvants.

Figure 22 - Immunogenicity of PE in bivalent formulations PD-PEPilA and trivalent PD-PEPilA-UspA2 with AS01E.

Figure 23 - Immunogenicity of PilA in bivalent formulations PD-PEPilA and trivalent PE-PilA-UspA2 with AS01E.

Figure 24 - Immunogenicity of PD in bivalent formulations PD-PEPilA and trivalent PE-PilA-UspA2 with AS01E.

Figure 25 - Effect of the tetravalent vaccine formulation PD / PEPilA / UspA2 / As01E on mouse lungs presensitized with M. cat. heat inactivated - Perivascularity and perbronchiolitis in PBS immunized mice.

Figure 26 - Effect of the tetravalent vaccine formulation PD / PEPilA / UspA2 / As01E on mouse lungs presensitized with M. cat. heat inactivated - Day 2 after immunization.

Figure 27 - Effect of the tetravalent vaccine formulation PD / PEPilA / UspA2 / As01E on mouse lungs presensitized with M. cat. heat inactivated - Day 7 after immunization.

Figure 28 - Effect of the tetravalent vaccine formulation PD / PEPilA / UspA2 / As01E on mouse lungs presensitized with M. cat. heat inactivated - Day 14 after immunization.

Figure 29 - Effect of the tetravalent vaccine formulation PD / PEPilA / UspA2 / As01E on mouse lungs presensitized with M. cat. inactivated by heat - Detailed results.

Figure 30 - Responses to CD4 T lymphocytes from the lungs after vaccination during restimulation with M. cat. Toilet. CD4 lymphocytes of the lungs expressing IL17. Restimulated with M. cat. whole cells (WC) inactivated with heat or medium.

Figure 31 - Responses to CD4 T lymphocytes from the lungs after vaccination during restimulation with M. cat. Toilet. CD4 lymphocytes of the lungs expressing TNFα. Restimulated with M. cat. whole cells (WC) inactivated with heat or medium.

Figure 32 - Responses to CD4 T lymphocytes from the lungs after vaccination during restimulation with M. cat. Toilet. CD4 lymphocytes of the lungs expressing IFNy. Restimulated with M. cat with whole cells (WC) inactivated with heat or medium.

Figure 33 - Responses to CD4 T lymphocytes from the lungs after vaccination during restimulation with M. cat. Toilet.

CD4 lymphocytes of the lungs expressing IL13. Restimulated with M. cat with whole cells (WC) inactivated with heat or medium.

Figure 34 - Responses in CD4 T cells of the lungs after challenge during restimulation with M. cat. Toilet. CD4 lymphocytes of the lungs expressing IL17. Restimulated with M. cat with whole cells (WC) inactivated with heat or medium.

Figure 35 - Responses to CD4 T lymphocytes from the lungs after challenge during restimulation with M. cat. Toilet. CD4 lymphocytes of the lungs expressing TNFα. Restimulated with M. cat with whole cells (WC) inactivated with heat or medium.

Figure 36 - Responses to CD4 T cells of the lungs after challenge during restimulation with M. cat. Toilet. CD4 lymphocytes of the lungs expressing IFNy. Restimulated with M. cat with whole cells (WC) inactivated with heat or medium.

Figure 37 - Responses to CD4 T cells of the lungs after challenge during restimulation with M. cat. Toilet. CD4 lymphocytes of the lungs expressing IL13. Restimulated with M. cat with whole cells (WC) inactivated with heat or medium.

Detailed description of the invention

Unless otherwise explained or defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. For example, definitions of common terms in molecular biology can be found in Benjamin Lewin, Gen. V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The terms singular "one", "one", "the" and "the" include plural articles unless the context clearly indicates otherwise. Similarly, the term "or" is meant to include "and" unless the context clearly indicates otherwise. It should further be understood that all base sizes or amino acid sizes, and all molecular weight or molecular weight values, given for the nucleic acids or polypeptides are approximate, and are provided for the description. In addition, the numerical limitations given with respect to the concentrations or levels of a substance, such as an antigen, may be approximate. Thus, when a concentration is indicated as being (for example) approximately 200 μg, the concentration is expected to include values slightly greater than or slightly less than ("about" or "~") 200 μg.

Although methods and materials similar or equivalent to those described herein may be used in the practice or analysis of this disclosure, suitable methods and materials are described below.

The term "includes" means "includes". Thus, unless the context requires otherwise, the term "includes", and variations such as "include" and "including" will be understood to imply the inclusion of a specified compound or composition (e.g. nucleic acid, polypeptide, antigen) or a step, or a group of compounds or steps, but not the exclusion of other compounds, compositions, steps, or groups thereof. The abbreviation "e.g." is derived from the Latin exempli gratia, and is used here to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example".

In order to facilitate the description of the various embodiments of this disclosure, the following explanations of the terms are provided. Additional terms and explanations are provided in the context of this disclosure.

A "subject" as used herein is a mammal, including humans, non-human primates, and non-primate mammals such as members of the rodent genus (including, but not limited to, mice and rats ) and members of the lagomorph order (including, but not limited to, rabbits).

As used herein, "UspA2" means Moraxella catarrhalis ubiquitous surface protein A2. UspA2 can consist of or comprise the amino acid sequence of SEQ ID NO: 1 ATCC 25238. MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNETLKGLITN SIENTNNITKNKADIQALENNVVEELFNLSG RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK VNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRV NPNLAFKAGAAINTSGNKKGSYNIGVNYEF (SEQ ID NO: 1) and sequences having at least or exactly 63%, 66%, 70%, 72%, 74%, 75%, 77%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity, over the entire length, with SEQ ID NO: 1. The comparison ison of 38 sequences of UspA2 from Moraxella catarrhalis (Table 1, SEQ ID NO: SEQ ID NO: 38) demonstrated approximately 63% at approximately 100% identity with UspA2 as represented by SEQ ID NO: 1.

UspA2 as described by SEQ ID NO: 1 contains a signal peptide (e.g. amino acids 1 to 29 of SEQ ID NO: 1), a laminin binding domain (e.g., amino acids 30 to 177 of SEQ ID NO: 1), a fibronectin binding domain (e.g., amino acids 165 to 318 of SEQ ID NO: 1) (Tan et al., JID 192: 1029-38 (2005)), a binding domain at C3 (e.g., amino acids 30 to 539 of SEQ ID NO: 1 (WO 2007/018463), or a fragment of amino acids 30 to 539 of SEQ ID NO: 1, for example, amino acids 165 to 318 of SEQ ID NO: 1 (Hallström T et al J. Immunol 186: 3120-3129 (2011)), an amphipathic helix (e.g., amino acids 519 to 564 of SEQ ID NO: 1 or amino acids 520 at 559 of SEQ ID NO: 1, identified using different prediction methods) and a C-terminal anchor domain (e.g., amino acids 576 to 630 of SEQ ID NO: 1 (Brooks et al., Infection & Immunity, 76 (11) , 5330-5340 (2008)).

Amino acid differences of UspA2 have been described for various species of Moraxella catarrhalis. See, for example, J Bacteriology 181 (13): 4026-34 (1999), Infection and Immunity 76 (11): 5330-40 (2008) and PLoS One 7 (9): e45452 (2012).

UspA2 may consist of or comprise an amino acid sequence that differs from SEQ ID NO: 1 at any one or more amino acids selected from the group consisting of: AA (amino acid) 30 to 298, AA 299 at 302, AA 303 to 333, AA 334 to 339, AA 349, AA 352 to 354, AA 368 to 403, AA 441, AA 451 to 471, AA 472, AA 474 to 483, AA 487, AA 490, AA 493 , AA 529, AA 532 or AA 543. UspA2 may consist of or comprise an amino acid sequence that differs from SEQ ID NO: 1 in that it contains at least one amino acid insert compared to SEQ ID NO UspA2 may consist of or comprise an amino acid sequence which differs from SEQ ID NO: 1 at any of the amino acid differences in SEQ ID NO: 2 to SEQ ID NO: 38. For example, SEQ ID NO: 1 may contain K instead of Q at amino acid 70, Q instead of G at amino acid 135 and / or D instead of N at of the amino acid 216.

Table 1 Amino acid sequences of UspA2 from 38 strains of Moraxella catarrhalis (SEQ ID NO: SEQ ID NO: 38)

UspA2 may be UspA2 of the strain of M. catarrhalis ATCC (a registered trademark in the United States) 25238 ™, American 2933, American 2912, American 2908, Finnish 307, Finnish 353, Finnish 358, Finnish 216, Dutch H2, Dutch F10 , Norwegian 1, Norwegian 13, Norwegian 20, Norwegian 25, Norwegian 27, Norwegian 36, BC5SV, Norwegian 14, Norwegian 3, Finish 414, Japanese Z7476, Belgium Z7530, German Z8063, American 012E, Greek MC317, American V1122, American P44 , American V1171, American TTA24, American 035E, American SP12-6, American SP12-5, Swedish BC5, American 7169, Finnish FIN2344, American V1118, American V1145 or American V1156. UspA2 may be UspA2 as represented by any of SEQ ID NO: 1 to SEQ ID NO: 38. UspA2 may be UspA2 from another source that corresponds to the UspA2 sequence of any of SEQ ID NO : 1 to SEQ ID NO: 38. Corresponding sequences of UspA2 can be determined by those skilled in the art using various algorithms. For example, the Gap program or the Needle program may be used to determine the UspA2 sequences corresponding to any one of SEQ ID NO: 1 to SEQ ID NO: 38.

UspA2 may be a sequence having at least 95% identity, over the entire length, with any one of SEQ ID NO: SEQ ID NO: 38 therein.

The immunogenic fragments of UspA2 comprise immunogenic fragments of at least 450 consecutive amino acids of SEQ ID NO: 1, 490 consecutive amino acids of SEQ ID NO: 1 (e.g., UspA2 fragment of MC-004 or MC-005 ), 511 consecutive amino acids of SEQ ID NO: 1 (for example, UspA2 fragment of construct MC-001, MC-002, MC-003 or MC-004), 534 consecutive amino acids of SEQ ID NO: 1 (e.g., UspA2 fragment of MC-009 or MC-011) or 535 consecutive amino acids of SEQ ID NO: 1 (e.g., UspA2 fragment of MC-007, MC-008 or MC-010). Immunogenic fragments can elicit antibodies that can bind SEQ ID NO: 1.

The immunogenic fragments of UspA2 may comprise immunogenic fragments of at least 450, 490, 511, 534 or 535 consecutive amino acids of any of SEQ ID NO: SEQ ID NO: 38. The immunogenic fragments of UspA2 may comprise immunogenic fragments of UspA2 of any of SEQ ID NO: 2 to SEQ ID NO: 38 which correspond to the UspA2 fragment of SEQ ID NO: 1 in any of the UspA2 constructs MC-001, MC-002 , MC-003, MC-004, MC-005, MC-006, MC-007, MC-008, MC-009, MC-010 or MC-011. The immunogenic fragments can trigger antibodies that can bind the full length sequence from which the fragment is derived.

Alignments between polypeptide pairs can be calculated by various programs. For example, the EMBOSS Needle program (free software, EMBOSS: The European Molecular Biology Open Software Suite (2000), Trends in Genetics 16 (6): 276-277) and the Gap program of the GCG software package (a registered trademark at USA) (Accelrys Inc.) can be used.

The Gap and Needle programs are an implementation of the Needleman-Wunsch algorithm described in: Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453. These programs frequently use the BLOSUM62 scoring matrix (Steven Henikoft and Jorja G. Henikoft (1992), "Amino acid substitution matrices from protein blocks"), Proc. Natl. Acad. Sci. USA 89 (Biochemistry): 10915-10919) with opening and gap extension penalties, respectively, of 8 and 2. Sometimes, the PAM250 scoring matrix (Dayhoft et al., (1978), "A model (3) MO Dayhoft (ed.), 345-352, National Biomedical Research Foundation, Washington) is also used.

The scoring matrices describe by numbers the tendency of each amino acid to mutate into another, or to be conserved. These numbers are usually calculated from statistics of mutations observed in faithful alignments in pairs or multiples, or even in fragments of multiple alignments. Generally, in these tables, if a high positive number is associated with a pair of identical amino acids, this indicates that this residue has a low tendency to mutation. In contrast, a high positive number associated with a different amino acid pair indicates a high tendency for mutation between these two amino acids. And this is called a "conservative substitution".

Looking at a pairwise alignment, one can observe identical aligned residues ("identities") between the two sequences. A percentage of identity can be calculated by multiplying by 100 (1) the quotient between the number of identities and the length of the alignment (for example, in the result of the Needle program), or (2) the quotient between the number of identities and the length of the longest sequence, or (3) the quotient between the number of identities and the length of the shortest sequence, or (4) the quotient between the number of identities and the number of aligned residues (for example, in the result of the Gap program).

The identity percentage of table 8 has been calculated according to the definition (3) of the preceding paragraph, using the pair alignments calculated by the Gap software.

As used herein, "adjuvant" means a compound or substance that, when administered to a subject in conjunction with a vaccine, immunotherapeutic agent, or other composition containing an antigen or immunogen, increases or amplifies the subject's immune response to the administered antigen or immunogen (compared to the immune response that would be achieved in the absence of the adjuvant). This should be distinguished from the "adjuvant therapy" defined by the National Cancer Institute of the United States Institutes of Health in the context of cancer treatment as an additional treatment given after primary treatment, to lower the risk of cancer recurring . The invention further provides proteins of formula (I) containing conservative amino acid substitutions. For example, the proteins of formula (I) may contain a conservative substitution of any amino acid of Moraxella catarrhalis UspA2 as described in any of the sequences presented herein (for example, any sequence of UspA2 represented by SEQ ID NO: 1 to SEQ ID NO: 38).

As used herein, "signal peptide" refers to a short polypeptide (less than 60 amino acids, e.g., 3 to 60 amino acids) present on precursor proteins (usually at the N-terminus), and which is usually absent from the mature protein. The signal peptide (sp) is generally rich in hydrophobic amino acids. The signal peptide directs the transport and / or secretion of the translated protein through the membrane. Signal peptides may also be referred to as targeting signals, transit peptides, location signals, or signal sequences. For example, the signal sequence may be a cotranslational or posttranslational signal peptide.

A heterologous signal peptide can be cleaved from a protein construct by peptide signal peptidases during or after transport or secretion of the protein. For example, the peptide signal peptidase is a peptide peptidase I peptide. A "heterologous" signal peptide is a signal peptide that is not associated with the protein as it exists in nature.

As used herein, "treatment" means preventing the occurrence of symptoms of the condition or disease in a subject, preventing the recurrence of symptoms of the condition or disease in a subject, the delay in the reappearance of symptoms of the condition or disease in a subject, decrease in the severity or frequency of symptoms of the condition or disease in a subject, slowdown or elimination of progression of the condition and the partial or total elimination of the symptoms of the disease or condition in a subject.

As used herein, "possibly" means that the event / events described hereinafter may / may not appear, and includes both the event / events that appear / appear and the events that occur. do not appear. Otitis media is a major cause of morbidity in 8 0% of all children under 3 years of age. (Expert Rev. Vaccines 5: 517-534 (2006)). More than 90% of children develop otitis media before the age of 7 (Current Opinion in

Investigational Drugs 4: 953-958 (2003)). In 2000, there were 16 million visits to physicians in private practice for otitis media in the United States and approximately 13 million prescriptions of antibacterials were delivered. (Pediatrics 113: 1451-1465 (2004)). In European countries, reported rates of acute otitis media range from 0.125 to 1.24 per child / year. (Expert Review of Vaccines 8: 1479-1500 (2009)). Otitis media is an expensive infection and the most common reason is that children get antibiotics. (Current Infectious Disease Reports 11: 177-182 (2009)). Bacteria are responsible for approximately 70% of cases of acute otitis media, with Streptococcus pneumoniae, nontypeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis predominating as causative agents (Expert Review of Vaccines 5: 517-534 (2006)) . A subset of children experiences recurrent and chronic otitis media and these children prone to otitis have prolonged middle ear effusions associated with hearing loss and speech development delays. and language. (Current Infectious Disease Reports 11: 177-182 (2009)). Recent antibiotic pressure and vaccination with pneumococcal conjugate vaccine resulted in Haemophilus influenzae and Moraxella catarrhalis producing ß-lactamase as the major causative organisms for acute otitis media in North America, followed by Streptococcus pneumoniae (Pediatric). Clin N Am 60 (2013) 391-407).

Since otitis media is a multifactorial disease, the feasibility of preventing otitis media using a vaccination strategy has been questioned. (Current Infectious Disease Reports 11: 177-182 (2009)).

The chinchilla model is a robust animal model validated by otitis media and its prevention (Expert Review of

Vaccines 8: 1063-1082 (2009)). While the chinchilla model may mimic the natural evolution of human infection, others have suggested that the results in the chinchilla model may vary from laboratory to laboratory. (Current Opinion in Investigational Drugs 4: 953-958 (2003)).

Various other rodents have also been used for the induction of otitis media and are summarized in Vaccine 26: 1501-1524 (2008). The murine animal model is often studied in otitis media research.

The presence of bactericidal antibodies is associated with protection against otitis media due to nontypable H. influenzae. (Current Opinion in Infectious Disease 16: 129-134 (2003)). However, an immune response does not need to be bactericidal to be effective against NTHi. Antibodies that simply react with the surface adhesins of NTHi can reduce or eliminate otitis media in chinchilla. (Current Opinion in Investigational Drugs 4: 953-958 (2003)).

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease of the lungs and a major cause of morbidity and mortality worldwide. Approximately one in 20 people in 2005 in the United States had COPD as their underlying cause. (Drugs and Aging 26: 985-999 (2009)). It is expected that by 2020, COPD will be the fifth leading cause of disability-adjusted life years, chronic disabling diseases, and the third leading cause of death (Lancet 349 1498-1504 (1997)). The evolution of COPD is characterized by a gradual worsening of airflow limitation and a decline in lung function. COPD can be complicated by frequent and recurrent acute exacerbations (AEs), which are associated with huge health expenditures and high morbidity. (Proceedings of the American Thoracic Society 4: 554-564 (2007)). One study suggests that approximately 50% of acute exacerbations of symptoms in COPD are caused by nontypable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. (Drugs and Aging 26: 985-999 (2009)). Haemophilus influenzae (H. influenzae) is found in 20 to 30% of COPD exacerbations; Streptococcus pneumoniae, in 10 to 15% of exacerbations of COPD; and Moraxella catarrhalis, in 10 to 15% of COPD exacerbations. (New England Journal of Medicine 359: 2355-2365 (2008)). Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis have been shown to be the primary pathogens in acute exacerbations of bronchitis in Hong Kong, South Korea, and the Philippines, while Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. . constitute a large proportion of pathogens in other Asian countries / regions including Indonesia, Thailand, Malaysia and Taiwan (Respirology, (2011) 16, 532-539, doi: 10.1111 / j.1440.1843.2011.01943.x). In Bangladesh, 20% of COPD patients had a positive sputum culture for Pseudomonas, Klebsiella, Streptococcus pneumoniae and Haemophilus influenzae, while 65% of patients with EABPCO (acute exacerbations of COPD) had positive cultures for Pseudomonas, Klebsiella, Acinetobacter, Enterobacter, Moraxella catarrhalis and their combinations. (Mymensingh Medical Journal 19: 576-585 (2010)). However, it has been suggested that the two most important measures to prevent exacerbation of COPD are active immunizations and chronic maintenance of pharmacotherapy. (Proceedings of the American Thoracic Society 4: 554-564 (2007)).

Outpatient pneumonia (HAP) has been described as the leading cause of death from infectious diseases and the sixth-largest cause of death in the United States. Moraxella catarrhalis is one of the pathogens associated with HEP in North America (Clin Chest Med 26 (2005) 37-55) and is one of the pathogens associated with moderate to severe hospital-acquired pneumonia in Japan ( J Infect Chemother, Nov. 2014, 20. S1341-321X (14) 00396-1, doi: 10.1016 / j.jiac.2014.11.006. [In-line delivery before printing]).

There is a need for effective vaccines against M. catarrhalis.

The present invention relates to proteins of formula (I).

wherein: A represents UspA2 of Moraxella catarrhalis or an immunogenic fragment thereof;

Ri represents an amino acid; m is 0 or 2; B represents a histidine; and n is 0, 1, 2, 3, 4, 5 or 6.

In a particular embodiment, Ri and m are defined where (Ri) m represents AS (alanine serine). In another embodiment, R 1 represents non-native amino acids.

In one embodiment, the proteins of formula (I) and the proteins of the invention are defined where m is 0. In one embodiment, when m is 0, n is 2. In another embodiment of the invention, the invention, when m is 0, n is not 0.

In one embodiment, m is 2.

In a particular embodiment, n is selected from the group consisting of 1, 2 and 6. In another embodiment, n is selected from the group consisting of 2 and 6. In a particular embodiment, n is 2 In another embodiment, n is 6.

In one embodiment, n is selected from the group consisting of 0, 1, 2 and 6, or any subset thereof.

In one embodiment, n is 0. In another embodiment, when n is 0, m is 2.

In one embodiment, n is 1. In one embodiment, n is 3. In one embodiment, n is 4. In one embodiment, n is 5.

In one embodiment, the proteins of formula (I) further contain a methionine (M) at the amino terminus; a protein with the following formula: methionine - A - (Ri) m (B) n · These are included in the proteins of the invention. In a particular embodiment, when m is 0 and n is 0, the proteins of formula (I) and the proteins of the invention are non-native proteins.

In one embodiment, the proteins of formula (I) and the proteins of the invention are non-native proteins.

In one embodiment, the proteins of formula (I) are defined where A represents UspA2 of M. catarrhalis. In another embodiment, the proteins of formula (I) are defined wherein A represents UspA2 as represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38 or any subset of SEQ ID NO: 1 to SEQ ID NO: 38. In another embodiment, the proteins of formula (I) are defined where A represents UspA2, where UspA2 is at least the same as 63%, 66%, 70%, 72%, 74%, 75%, 77%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% over the entire length, with SEQ ID NO: 1. In another embodiment, the proteins of formula (I) are defined where A is UspA2, where UspA2 is approximately 75% to 100% identical with the acid sequence Amines of UspA2 represented by SEQ ID NO: 1. In another embodiment, A represents UspA2 where UspA2 is approximately 90% to 100% identical with the amino acid sequence of UspA2 represented by SEQ ID NO: 1. another embodiment, A represents UspA2, where UspA2 is at least 95% identical with the amino acid sequence of UspA2 represented by SEQ ID NO: 1. In another embodiment, the proteins of formula (I) are defined where A is UspA2, where UspA2 is approximately 75% to 100% identical to the UspA2 amino acid sequence represented by any SEQ ID NO: 1 to SEQ ID NO: 38. In another embodiment, A represents UspA2, where UspA2 is approximately 90% to 100% identical to the amino acid sequence of UspA2 represented by one of Any of SEQ ID NO: 1 to SEQ ID NO: 38. In a further embodiment, A represents UspA2, where UspA2 is at least 95% identical to the amino acid sequence of UspA2 represented by any one of SEQ ID NO: SEQ ID NO: 38. In a particular embodiment, A represents UspA2 having the amino acid sequence represented by SEQ ID NO: 1.

In another embodiment, the proteins of formula (I) are defined where A represents an immunogenic fragment of UspA2 of M. catarrhalis. In another embodiment, A is an immunogenic fragment of UspA2 where UspA2 has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO : 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38 or any subset of SEQ ID NO: 1 to SEQ ID NO: 38. In another embodiment, A is an immunogenic fragment of UspA2, where UspA2 is approximately 75% to 100% identical. with the amino acid sequence represented by SEQ ID NO: 1. In another embodiment, A is an immunogenic fragment of UspA2, where UspA2 is approximately 90% to 100% identical with SEQ ID NO: 1. In another embodiment, A is a fragment immunogen of UspA2, where UspA2 is at least 95% identical with SEQ ID NO: 1. In another embodiment, A is an immunogenic fragment of UspA2, where UspA2 is approximately 75% to 100% identical with the sequence of amino acid represented by any one of SEQ ID NO: 1 to SEQ ID NO: 38. In another embodiment, A is an immunogenic fragment of UspA2, where UspA2 is approximately 90% to 100% identical with any one of SEQ ID NO: 1 to SEQ ID NO: 38. In a further embodiment, A is an immunogenic fragment of UspA2, where UspA2 is at least 95% identical with any of SEQ ID NO: SEQ ID NO: 38. In a particular embodiment, A is an immunogenic fragment of UspA2, where UspA2 has the amino acid sequence represented by SEQ ID NO: 1.

In another embodiment, A is an immunogenic fragment of UspA2 from M. catarrhalis selected from the group consisting of amino acids 30 to 540 of SEQ ID NO: 1 (SEQ ID NO: 39), amino acids 31 to 540 of SEQ ID NO: 1 (SEQ ID NO: 40), amino acids 30 to 519 of SEQ ID NO: 1 (SEQ ID NO: 41), amino acids 30 to 564 of SEQ ID NO: 1 (SEQ ID NO: 42) and amino acids 31 to 564 of SEQ ID NO: 1 (SEQ ID NO: 43). More specifically, in a mode of

embodiment, A represents SEQ ID NO: 43, amino acids 31 to 564 of SEQ ID NO: 1. In a further embodiment, A is SEQ ID NO: 42, amino acids 30 to 564 of SEQ ID NO: 1 In another embodiment, A is an immunogenic fragment of UspA2 from M. catarrhalis selected from the group consisting of amino acids 30 to 540 of SEQ ID NO: 1 (SEQ ID NO: 39), amino acids 31 to 540 of SEQ ID NO: 1 (SEQ ID NO: 40) and amino acids 30 to 519 of SEQ ID NO: 1 (SEQ ID NO: 41). In another embodiment, A is an immunogenic fragment of UspA2 exhibiting at least 52% (American 2908), 55% (Norwegian 25), 57% (Japanese Z7476), 62% (Finnish FIN2344), 64% (American 2912). ), 69% (American P44), 73% (American 7169), 76% (Norwegian 27), 81% (American V1145), 88% (German Z8063) or 100% (Swedish BC5) identity with SEQ ID NO : 39. In another embodiment, A is an immunogenic fragment of UspA2 having at least 52% (American 2908), 57% (Dutch F10), 62% (American 2933), 65% (Greek MC317), 67% (American V1122), 70% (American P44), 73% (American 7169), 76% (Norwegian 3), 81% (German Z8063), 100% (Swedish BC5) identity with SEQ ID NO: 43.

In another embodiment, A is an immunogenic fragment of UspA2 from M. catarrhalis of SEQ ID NO: 2 to SEQ ID NO: 38 where the fragment comprises amino acids that align with SEQ's amino acids 30 to 540. ID NO: 1 (SEQ ID NO: 39), amino acids 31 to 540 of SEQ ID NO: 1 (SEQ ID NO: 40), amino acids 30 to 519 of SEQ ID NO: 1 (SEQ ID NO: 41 ), amino acids 30 to 564 of SEQ ID NO: 1 (SEQ ID NO: 42) or amino acids 31 to 564 of SEQ ID NO: 1 (SEQ ID NO: 43). In one embodiment, the Gap program (of the GCG package), or the Needle program (of the EMBOSS package), implementing the Needleman-Wunsch algorithm, may be used to align the sequences.

UspA2 - SEQ ID NO: 1

MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNETLKGLITN SIENTNNITKNKADIQALENNVVEELFNLSG RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK VNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRV N PNLAFKAGAAINTS GNKKGS YNIGVNYEF

Amino acids 30 to 540 of UspA2 of SEQ ID NO: 1, SEQ ID NO: 39

QAKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENTNNITKNKADIQALENNVVEELFNLS G RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK

Amino acids 31 to 540 of UspA2 of SEQ ID NO: 1, SEQ ID NO: 40

AKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENTNNITKNKADIQALENNVVEELFNLS G RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK

Amino acids 30 to 519 of UspA2 of SEQ ID NO: 1, SEQ ID NO: 41

QAKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENTNNITKNKADIQALENNVVEELFNL S G RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKS

Amino acids 30 to 564 of UspA2 of SEQ ID NO: 1, SEQ ID NO: 42

QAKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENTNNITKNKADIQALENNVVEELFNL S G RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK

VNAFDGRITALDSKVENGMAAQAA

Amino acids 31 to 564 of UspA2 of SEQ ID NO: 1, SEQ ID NO: 43

AKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENTNNITKNKADIQALENNVVEELFNLS G RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK VNAFDGRITALDSKVENGMAAQAA

In another embodiment, A is an immunogenic fragment of UspA2 from M. catarrhalis which differs from SEQ ID NO: 1 in one or more of the following amino acids: AA (amino acid) 30 to 298, AA 299 to 302, AA 303 to 333, AA 334 to 339, AA 349, AA 352 to 354, AA 368 to 403, AA 441, AA 451 to 471, AA 472, AA 474 to 483, AA 487, AA 490, AA 493, AA 529, AA 532 or AA 543. In another embodiment, A is an immunogenic fragment of UspA2 from M. catarrhalis which differs from SEQ ID NO: 1 in that it contains at least one amino acid insertion compared to SEQ ID NO: 1.

In another embodiment, A is an immunogenic fragment of UspA2 that contains a laminin binding domain and a fibronectin binding domain.

In a further embodiment, A is an immunogenic fragment of UspA2 that contains a laminin binding domain, a fibronectin binding domain, and a C3 binding domain.

In another embodiment, A is an immunogenic fragment of UspA2 that contains a laminin binding domain, a fibronectin binding domain, a C3 binding domain, and an amphipathic helix.

The laminin binding domain, the fibronectin binding domain, the C3 binding domain or the amphipathic helix may be as defined for SEQ ID NO: 1 or may be the sequence corresponding to any one of SEQ ID NO: 2 to SEQ ID NO: 38.

The proteins of formula (I) and the proteins of the invention are useful as immunogens in subjects such as mammals, particularly humans. In particular, the proteins of formula (I) and the proteins of the invention are useful in inducing an immune response against M. catarrhalis in subjects, particularly humans. The proteins of formula (I) and the proteins of the invention are useful in the treatment or prevention of an infection or disease due to M. catarrhalis. More specifically, the proteins of formula (I) and the proteins of the invention are useful in the treatment or prevention of otitis media and / or COPD and / or EABPCO and / or pneumonia.

The present invention relates to immunogenic compositions comprising UspA2 of M. catarrhalis or an immunogenic fragment thereof. The present invention also relates to vaccines comprising such immunogenic compositions and their therapeutic uses. The immunogenic compositions and vaccines of the present invention are useful in the treatment or prevention of M. catarrhalis infection or disease. More specifically, the immunogenic compositions and vaccines described herein are useful in the treatment or prevention of otitis media and / or COPD and / or EABPCO and / or pneumonia.

In one embodiment, the immunogenic composition comprises UspA2 from M. catarrhalis. UspA2 can be any of SEQ ID NO: 1 to SEQ ID NO: 38 or a sequence of UspA2 at least 75% identical, 80%, 85%, 90%, 95%, 96%, 97%, 98 % or 99% with any of SEQ ID NO: 1 to SEQ ID NO: 38. UspA2 may be also a sequence of UspA2 at least 63% identical (American 2908), 66% (Japanese Z7476), 70% (Dutch F10), 72% (Finnish 358), 74% (American P44), 77% (Finnish 307), 80% (Norwegian 3), 84% (American V1145), 90% (German Z8063) or 100% ( Swedish BC5) with that of SEQ ID NO: 1.

In another embodiment, the immunogenic composition comprises an immunogenic fragment of UspA2. The immunogenic fragment of UspA2 can be SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43, or a sequence having at least 90%, 95%, 96 %, 97%, 98%, 99% sequence identity with any of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43 The immunogenic fragment of UspA2 can be a sequence of UspA2 at least identical to 52% (American 2908), 55% (Norwegian 25), 57% (Japanese Z7476), 62% (Finnish FIN2344), 64% (American 2912) , 69% (American P44), 73% (American 7169), 76% (Norwegian 27), 81% (American V1145), 88% (German Z8063) or 100% (Swedish BC5) with SEQ ID NO: 39. immunogenic fragment of UspA2 may be also a sequence of UspA2 at least identical to 52% (American 2908), 57% (Dutch F10), 62% (American 2933), 65% (Greek MC317), 67% (American V1122), 70% (American P44), 73% (American 7169), 76% (Norwegian 3), 81% (German Z8063), 100% (Swedish BC5) with SE Q ID NO: 43. Amino acid differences have been described in UspA2 from various species of Moraxella catarrhalis.

UspA2 contains a laminin binding domain (e.g., amino acids 30 to 177 of SEQ ID NO: 1, SEQ ID NO: 44). In one embodiment, the UspA2 fragment comprises the laminin binding region of SEQ ID NO: 1. In a further embodiment, the UspA2 fragment comprises the laminin binding region of SEQ ID NO: 2. at SEQ ID NO: 38.

Amino acids 30 to 177 of SEQ ID NO: 1, SEQ ID NO: 44: QAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDV GWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQR NLVNGFEIEKNKDAIAKNNESIED

UspA2 contains a fibronectin binding domain (e.g., amino acids 165 to 318 of SEQ ID NO: 1, SEQ ID NO: 45). In one embodiment, the fragment of

UspA2 comprises the fibronectin binding region of SEQ ID NO: 1. In a further embodiment, the UspA2 fragment comprises the fibronectin binding region of any of SEQ ID NO: 2 to SEQ ID NO The fibronectin binding domain of SEQ ID NO: 45 also has C3 binding properties.

Amino acids 165 to 318 of SEQ ID NO: 1, SEQ ID NO: 45:

KDAIAKNNESIEDLYD

FGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSG RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQK

UspA2 contains a complement component 3 (C3) binding domain (e.g., amino acids 30 to 539 of SEQ ID NO: 1, SEQ ID NO: 46, or amino acids 165 to 318 of SEQ ID NO: 1 , SEQ ID NO: 45). In one embodiment, the UspA2 fragment comprises the C3 binding region of SEQ ID NO: 1. In a further embodiment, the UspA2 fragment comprises a C3 binding domain of any one of SEQ IDs. NO: 2 to SEQ ID NO: 38.

Amino acids 30 to 539 of SEQ ID NO: 1, SEQ ID NO: 46:

QAKNDITLEDLPYLIKKIDQNELEADIGDIT ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD FGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENTNNITKNKADIQALENNVVEELFNLS G RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDT

UspA2 contains an amphipathic helix (e.g., amino acids 519 to 564 of SEQ ID NO: 1 or amino acids 520 to 559 of SEQ ID NO: 1). In one embodiment, the UspA2 fragment comprises amino acids 519 to 564 of SEQ ID NO: 1. In another embodiment, the UspA2 fragment comprises amino acids 520 to 559 of SEQ ID NO: 1. In a further embodiment, the UspA2 fragment comprises an amphipathic helix of any one of SEQ ID NO: 2 to SEQ ID NO: 38.

In one embodiment, the immunogenic composition comprises a protein of formula (I) wherein A is an immunogenic fragment of UspA2 which comprises a laminin binding domain and a fibronectin binding domain.

In a further embodiment, the immunogenic composition comprises a protein of formula (I) wherein A is an immunogenic fragment of UspA2 which comprises a laminin binding domain, a fibronectin binding domain and a C3 binding domain. .

In another embodiment, the immunogenic composition comprises a protein of formula (I) wherein A is an immunogenic fragment of UspA2 which comprises a laminin binding domain, a fibronectin binding domain, a C3 binding domain and an amphipathic helix.

In another embodiment, the immunogenic composition comprises a protein as defined by formula (I). The immunogenic composition may contain, for example, a protein of formula (I) with an additional methionine at the amino terminus.

In one embodiment, the present immunogenic compositions may be administered with other antigens. For example, the present immunogenic composition may be administered with H. influenzae antigens. For example, the protein of formula (I) may be administered with H. influenzae protein D (PD). Protein D can be as described in WO 91/18926. The present immunogenic composition may be administered with H. influenzae protein E (PE) and pilin A (PilA). Protein E and pilin A may be as described in WO 2012/139225; whose contents are incorporated herein by reference. Protein E and pilin A may be in the form of a fusion protein.

In another embodiment, the immunogenic compositions of the invention may be administered with additional antigens from other bacterial species also known to cause otitis media, COPD, EABPCO or pneumonia.

The amount of the immunogenic composition that is required to achieve the desired therapeutic or biological effect will depend on a number of factors such as the use for which it is intended, the means of administration, the recipient and the type and the severity of the condition being treated, and it will ultimately be at the discretion of the attending physician or veterinarian. In general, it can be expected that a typical dose for treating a condition caused in whole or in part by M. catarrhalis in a human, for example, is in the range of about 0.001 mg. at 0.120 mg. More specifically, a typical dose for treating a condition caused in whole or in part by M. catarrhalis in a human may be in the range of about 0.003 mg to about 0.03 mg of protein. The present invention provides an immunogenic composition comprising a protein of formula (I) or a protein of the invention for use in the treatment or prevention of an ailment or disease caused in whole or in part by M. catarrhalis . The immunogenic composition may contain additional antigens; a typical dose for treating a condition caused in whole or in part by H. influenzae in a human may be in the range of about 0.005 mg to about 0.05 mg for each additional antigen. This dose may be administered as a single unit dose. Several unit doses can also be administered separately. For example, unit doses may be administered separately as separate sensitizing doses in the first year of life or as separate booster doses given at regular intervals (eg every 1, 5 or 10 years ). The present invention also provides an immunogenic composition comprising a protein of formula (I) or a protein of the invention for use in the treatment or prevention of a condition or disease caused in whole or in part by Moraxella catarrhalis in combination with at least one antigen from Haemophilus influenzae.

Formulations comprising the immunogenic compositions of the invention may be adapted for administration by a suitable route, for example, intramuscularly, sublingually, transcutaneously, intradermally or intranasally. Such formulations can be prepared by any method known from the state of the art.

The immunogenic compositions of the present invention may further comprise an adjuvant. When the term "adjuvant" is used in this specification, it refers to a substance that is administered together with the immunogenic composition to stimulate the immune response of the patient to the immunogenic component of the composition.

Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or alum, but it may also be a salt of calcium, magnesium, iron or zinc, or it may be may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derived saccharides, or polyphosphazenes. In one embodiment, the protein can be adsorbed on aluminum phosphate. In another embodiment, the protein may be adsorbed onto aluminum hydroxide. In a third embodiment, alum may be used as an adjunct.

Suitable adjuvant systems that favor a predominantly Th1 response include: non-toxic derivatives of lipid A, monophosphoryl lipid A (MPL) or a derivative thereof, particularly monophosphoryl lipid A 3-des-O- acylated (3D-MPL) (for preparation, see GB 2220211 A); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with either an aluminum salt (e.g., aluminum phosphate or aluminum hydroxide) an oil emulsion in water. In such combinations, the antigen and 3D-MPL are contained in the same particulate structures, allowing for more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further enhance the immunogenicity of an alum adsorbed antigen (Thoelen et al., Vaccine (1998) 16: 708-14, EP 689454-B1). AS01 is an adjuvant system containing MPL (3-0-deacyl-4'-monophosphoryl lipid A), QS21 (Quillaja saponaria Molina, fraction 21) Antigenics, New York, NY, USA) and liposomes. AS01B is an adjuvant system containing MPL, QS21 and liposomes (50 μg MPL and 50 μg QS21). AS01E is an adjuvant system containing MPL, QS21 and liposomes (25 μg MPL and 25 μg QS21). In one embodiment, the immunogenic composition or the vaccine comprises AS01. In another embodiment, the immunogenic composition or the vaccine comprises AS01B or AS01E. In a particular embodiment, the immunogenic composition or the vaccine comprises AS01E. AS02 is an adjuvant system containing MPL and QS21 in an oil / water emulsion. AS02V is an adjuvant system containing MPL and QS21 in an oil / water emulsion (50 μg MPL and 50 μg QS21). AS03 is an adjuvant system containing α-tocopherol and squalene in an oil / water emulsion (w / w). AS03a is an adjuvant system containing α-tocopherol and squalene in an o / w emulsion (11.86 mg tocopherol). AS03B is an adjuvant system containing α-tocopherol and squalene in an o / w emulsion (5.93 mg tocopherol). AS03C is an adjuvant system containing α-tocopherol and squalene in an o / w emulsion (2.97 mg tocopherol). In one embodiment, the immunogenic composition or the vaccine comprises AS03. AS04 is an adjuvant system containing MPL (50 μg MPL) adsorbed on an aluminum salt (500 μg Al3 +). In one embodiment, the immunogenic composition or the vaccine comprises AS04.

A system involving the use of QS21 and 3D-MPL is described in WO 94/00153. A composition in which QS21 is neutralized with cholesterol is described in WO 96/33739. An additional adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210. In one embodiment, the immunogenic composition further comprises a saponin, which may be QS21. The formulation may also include an oil-in-water emulsion and tocopherol (WO 95/17210). Oligonucleotides containing unmethylated CpGs (WO 96/02555) and other immunomodulatory oligonucleotides (WO 0226757 and WO 03507822) are also preferred inducers of a TH1 response and are suitable for use in the present invention.

Additional adjuvants are those selected from the group of metal salts, oil-in-water emulsions, Toll-type receptor agonists (in particular the Toll-type receptor agonist, the Toll-type receptor agonist). 3, Toll receptor agonist 4, Toll receptor agonist 7, Toll receptor agonist 8 and Toll receptor agonist 9), saponins or combinations thereof -this.

The present invention provides a process for preparing an immunogenic composition comprising combining a protein of formula (I) or a protein of the invention with an adjuvant.

The present invention further provides a vaccine containing an immunogenic composition of the invention and a pharmaceutically acceptable adjuvant.

Possible excipients include arginine, pluronic acid and / or polysorbate. In a preferred embodiment, polysorbate 80 (e.g., TWEEN (a registered trademark in the United States) 80) is used. In another embodiment, a final concentration of about 0.03% to about 0.06% is used. Specifically, a final concentration of about 0.03%, 0.04%, 0.05% or 0.06% polysorbate 80 (w / v) can be used.

The present invention provides a method for preparing an immunogenic composition or vaccine comprising combining a protein of formula (I) or a protein of the invention with a pharmaceutically acceptable excipient.

The present invention also provides nucleic acids encoding the proteins of the invention. The term "nucleic acid" refers to a polymeric form of nucleotides. The nucleotides may be ribonucleotides, deoxyribonucleotides, or modified forms of either ribonucleotides or deoxyribonucleotides. The term includes single and double forms of DNA. The nucleic acids are preferably substantially free of other nucleic acids.

The present invention provides a method for producing the nucleic acids of the invention. The nucleic acids of the invention can be prepared by methods known to those skilled in the art. For example, the nucleic acids of the invention may be synthesized in part or in whole. Nucleic acids can be prepared by digestion of longer amino acids or junction of shorter amino acids.

The present invention provides a method for treating or preventing otitis media. The method comprises administering to a subject in need thereof a therapeutically effective amount of a protein of formula (I) or a protein of the invention.

The present invention provides a method for the treatment or prevention of exacerbations in chronic obstructive pulmonary disease. Exacerbation of COPD can be an acute exacerbation. The method comprises administering to a subject in need thereof a therapeutically effective amount of the protein of formula (I) or a protein of the invention.

The present invention provides a method for the treatment or prevention of pneumonia. The method comprises administering to a subject in need thereof a therapeutically effective amount of the protein of formula (I) or a protein of the invention.

The present invention provides a pharmaceutical composition comprising a protein of formula (I) or a protein of the invention for use in the treatment or prevention of an ailment or disease caused in whole or in part by Moraxella catarrhalis. The pharmaceutical compositions may further comprise a pharmaceutically acceptable adjuvant.

The present invention provides the use of (a) proteins of formula (I) and proteins of the invention, (b) an immunogenic composition comprising a protein of formula (I) or a protein of the invention or ( c) a vaccine comprising (cl) a protein of formula (I) or a protein of the invention or (c2) an immunogenic composition comprising a protein of formula (I) or a protein of the invention for the manufacture of 'a medicinal product for the treatment or prevention of an infection or disease caused by M. catarrhalis.

The present invention provides the use of (a) proteins of formula (I) and proteins of the invention, (b) an immunogenic composition comprising a protein of formula (I) or a protein of the invention or ( c) a vaccine comprising (cl) a protein of formula (I) or a protein of the invention or (c2) an immunogenic composition comprising a protein of formula (I) or a protein of the invention for the manufacture of 'a medicine for the treatment or prevention of otitis media.

The present invention provides the use of (a) proteins of formula (I) and proteins of the invention, (b) an immunogenic composition comprising a protein of formula (I) or a protein of the invention or ( c) a vaccine comprising (cl) a protein of formula (I) or a protein of the invention or (c2) an immunogenic composition comprising a protein of formula (I) or a protein of the invention for the manufacture of 'a drug for the treatment or prevention of acute exacerbations of chronic obstructive pulmonary disease (COPD).

The present invention provides the use of (a) proteins of formula (I) and proteins of the invention, (b) an immunogenic composition comprising a protein of formula (I) or a protein of the invention or ( c) a vaccine comprising (cl) a protein of formula (I) or a protein of the invention or (c2) an immunogenic composition comprising a protein of formula (I) or a protein of the invention for the manufacture of 'a medicine for the treatment or prevention of pneumonia.

The following examples are intended only as an illustration and are not intended to limit the scope of the invention in any way.

In the examples, the following terms have the designated meaning: 6xhis = six histidines; xg = centrifugal force (number of gravities); AS = serine alanine; BSA = bovine serum albumin; ° C = degrees Celsius;

CaCl2 = calcium chloride; CD = circular dichroism; CHCl3 = chloroform; CH3CN = acetonitrile; CO2 = carbon dioxide;

Da = dalton; DNA = deoxyribonucleic acid; OD = dissolved oxygen; DSC = differential scanning calorimetry; EDTA = ethylene-diamine-tetraacetic acid; h = hour; H20 = water; H2O2 = hydrogen peroxide; HCDI = high cell density induction; HCl = hydrogen chloride;

His = his = histidine; IMAC = affinity chromatography with immobilized metals; IPTG = isopropyl-β-D-1-thiogalactopyranoside; kVolts = kilovolts; L = liter; LB = Luria-Bertani; LCDI = low cell density induction; MeOH = methanol; ml = milliliter;

NaCl = sodium chloride; rpm = revolutions per minute; min = minute; mM = millimolar; μρ = microgram; μΐ = microliter; PM = molecular weight; m / z = mass / charge;

NaCl = sodium chloride;

NaP04 = sodium phosphate; ng = nanograirane; NH4OH = ammonium hydroxide; nm = nanometer; D.O. = optical density; PBS = phosphate buffer solution; PCR = polymerase chain reaction; psi = pounds per square inch; PVDF = polyvinylidene difluoride; SDS-PAGE = polyacrylamide gel electrophoresis with sodium dodecyl sulfate; TFA = trifluoroacetic acid;

Tm = melting point;

Tmi = first melting point;

Tnp = second melting point; p / v = weight / volume.

Examples

Example 1 - Protein Constructs

Protein constructs were produced with different UspA2 fragments with and without additional amino acids. The following table describes the protein constructs made.

Table 2

Constructs of proteins containing UspA2 protein

A.A. = amino acid

The DNA and amino acid sequences for each protein construct listed in Table 2 are shown below: PROTEIN CONSTRUCTIONS SEQUENCES MC-001 (DNA) - SEQ ID NO: 52

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC

ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGCAAGCCATCATCA

TCACCACCACTAA MC-001 (Protein) - (M) (UspA2 amino acids 30 to 540) (ASHHHHHH) SEQ ID NO: 53

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLG TKVDGFDSRVTALDTKASHHHHHH MC-002 (DNA) - SEQ ID NO: 54

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA CAGAAT CCGA T T T GGCAGAACAGGGT GAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGAT TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGA CTGAAGCCATCGA CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAATAA MC-002 (protein) - (M) (amino acids 30-540 of UspA2) SEQ ID NO: 55

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED

VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ

RNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENT

NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN

VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE

DLAAYNELQD

AYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKN QADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIE KDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRV TALDTK SEQ ID NO: 56

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC

ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGA

TGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGAATGGCAGCACAAGCAGCACACT

AA SEQ ID NO: 57

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLG TKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAH TM-003 (DNA) - SEQ ID NO: 87

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC

ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAACACTAA MC-003 (Protein) - (M) (amino acids 30 to 540 of UspA2) (H) - SEQ ID NO: 88

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNE SIE DLYDFGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLG TKVDGFDSRVTALDTKH TM-004 (DNA) - SEQ ID NO: 58

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA CT GAC GAAAAAC CAGAACAC CCT GAT T GAAAAAGATAAAGAACAT GATAAAC T GAT CACCGC CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAACATCATTAA MC-004 (protein) - (M) (amino acids 30-540 of UspA2) (HH) SEQ ID NO : 59

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNE SIE DLYDFGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLG TKVDGFDSRVTALDTKHH TM-005 (DNA) - SEQ ID NO: 60

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCGCAAGCCATCATCAT

CACCACCACTAA MC-005 (Protein) - (M) (amino acids 30 to 519 of UspA2) (ASHHHHHH) SEQ ID NO: 61

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSASHHH HHH MC-O O 6 (DNA) - SEQ ID NO: 62

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCTAA MC-006 (Protein) - (M) (amino acids 30 to 519 of UspA2) SEQ ID NO: 63

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED

VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ

RNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENT

NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN

VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE

DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKS MC-007 (ADN) - SEQ ID NO: 64

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC

ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGA

TGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCAGCAA

GCCATCATCATCACCACCACTAA MC-007 (Protein) - (M) (amino acids 30 to 564 of UspA2) (ASHHHHHH) SEQ ID NO: 65

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLG TKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAASHHHHHH MC-008 (ADN) - SEQ ID NO: 66

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC

ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGA

TGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCACACC

ACTAA MC-008 (Protein) - (M) (30 to 564 from UspA2) (HH) SEQ ID NO: 67

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNE SIE DLYDFGHEVAE SIGEIHAHNEAQNE TLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLG TKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAHH TM-009 (DNA) - SEQ ID NO: 68

ATGGCGAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAA

CGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGT

ATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTG

GGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCA

GAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTG

TTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGT

AATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAG

CATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCAC

ATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAAT

AACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTT

TAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTT

ATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTT

GAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAA

TCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAAC

AGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGAT

CTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGC

ACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAAT

TACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCT

GAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTA

TGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCG

CAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTG

ACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAA

TAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATG

CAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACC

AAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGATGG

TCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCACACCACT

AA MC-O 9 (Protein) - (M) (31 to 564 from UspA2) (HH) SEQ ID NO: 69

MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDV GWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQR NLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTN NITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNV EEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIED LAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASS ENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETL TKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGT KVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAHH TM-010 (DNA) - SEQ ID NO: 70

ATGCAGGCCAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCA

GAACGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCC

AGTATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGAT

GTGGGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAA

TCAGAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATT

TTGTTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAG

CGTAATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGA

AAGCATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATG

CACATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACC

AATAACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACT

GTTTAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACA

TTTATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAAC

GTTGAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCA

GAATCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGA

AACAGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAA

GATCTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGA

CGCACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATG

AATTACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCT

TCTGAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATAT

CTATGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAA

GCGCAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACA

CTGACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGC

CAATAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAG

ATGCAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGC

ACCAAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGTTAATGCATTTGA

TGGTCGTATTACCGCTCTGGATAGTAAAGTTGAAAATGGTATGGCAGCACAGGCAGCATAA MC-010 (Protein) - (M) (amino acids 30 to 564 of UspA2) SEQ ID NO: 71

MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDED VGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQ RNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENT NNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKN VEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFET

LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLG TKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAA MC-011 (DNA) - SEQ ID NO: 72

ATGGCGAAAAATGATATTACCCTGGAAGATCTGCCGTATCTGATCAAAAAAATCGATCAGAA

CGAACTGGAAGCCGATATTGGTGATATTACCGCACTGGAAAAATATCTGGCACTGAGCCAGT

ATGGAAATATTCTGGCCCTGGAAGAACTGAATAAAGCTCTGGAAGAGCTGGATGAAGATGTG

GGTTGGAATCAGAATGATATCGCCAATCTGGAAGATGATGTTGAAACCCTGACCAAAAATCA

GAATGCACTGGCAGAACAGGGTGAAGCAATTAAAGAAGATCTGCAGGGTCTGGCAGATTTTG

TTGAAGGTCAGGAAGGCAAAATTCTGCAGAACGAAACCAGCATCAAAAAAAACACCCAGCGT

AATCTGGTGAATGGCTTTGAAATTGAAAAAAACAAAGATGCCATTGCCAAAAACAACGAAAG

CATTGAAGATCTGTATGATTTTGGTCATGAAGTTGCCGAAAGCATTGGTGAAATTCATGCAC

ATAACGAAGCACAGAATGAAACCCTGAAAGGTCTGATTACCAACAGCATCGAAAATACCAAT

AACATTACCAAAAACAAAGCAGATATTCAGGCGCTGGAAAATAATGTTGTGGAAGAACTGTT

TAATCTGAGCGGTCGTCTGATTGATCAGAAAGCCGATATCGATAATAACATTAACAACATTT

ATGAACTGGCACAGCAGCAGGATCAGCATAGCAGCGATATCAAAACCCTGAAAAAAAACGTT

GAAGAAGGTCTGCTGGAACTGTCTGGTCACCTGATCGATCAGAAAACTGATATTGCCCAGAA

TCAGGCAAATATTCAGGATCTGGCCACCTATAATGAACTGCAGGATCAGTATGCACAGAAAC

AGACCGAAGCAATTGATGCCCTGAATAAAGCGAGCAGCGAAAACACCCAGAATATCGAAGAT

CTGGCAGCATACAACGAACTGCAGGATGCCTATGCAAAACAGCAGACTGAAGCCATCGACGC

ACTGAACAAGGCAAGCTCTGAAAACACGCAGAACATTGAAGATCTGGCTGCCTATAATGAAT

TACAGGATGCGTATGCCAAACAGCAGACCGAAGCGATTGATGCGCTGAACAAAGCCTCTTCT

GAAAATACACAGAATATCGCCAAAAATCAGGCCGATATTGCCAACAATATCAATAATATCTA

TGAACTGGCCCAGCAGCAGGATCAGCACTCTTCTGATATCAAAACACTGGCAAAAGCAAGCG

CAGCAAATACCGATCGTATTGCGAAAAACAAAGCCGATGCAGATGCAAGCTTTGAAACACTG

ACGAAAAACCAGAACACCCTGATTGAAAAAGATAAAGAACATGATAAACTGATCACCGCCAA

TAAAACCGCAATTGATGCAAATAAAGCCAGCGCAGATACCAAATTTGCAGCAACCGCAGATG

CAATTACCAAAAATGGCAATGCCATCACCAAAAATGCCAAAAGCATTACCGATCTGGGCACC

AAAGTTGATGGTTTTGATAGCCGTGTGACCGCACTGGATACCAAAGCAAGCCATCATCATCA

CCACCACTAA MC-011 (Protein) - (M) (amino acids 31 to 540 of UspA2) (ASHHHHHH) SEQ ID NO: 73

MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDV GWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQR NLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTN NITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNV EEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIED LAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASS ENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETL TKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGT KVDGFDSRVTALDTKASHHHHHH

Vector Construction and Transformation DNA Sequence for UspA2 from ATCC Strain 25238 - SEQ ID NO: 74

ATGAAAACCATGAAACTTCTCCCTCTAAAAATCGCTGTAACCAGTGCCATGATTATTGGCTT

GGGTGCGGCATCTACTGCGAATGCGCAGGCTAAAAATGATATAACTTTAGAGGATTTACCAT

ATTTAATAAAAAAGATTGACCAAAATGAATTGGAAGCAGATATCGGAGATATTACTGCTCTT

GAAAAGTATCTAGCACTTAGCCAGTATGGCAATATTTTAGCTCTAGAAGAGCTCAACAAGGC

TCTAGAAGAGCTCGACGAGGATGTTGGATGGAATCAGAATGATATTGCAAACTTGGAAGATG

ATGTTGAAACGCTCACCAAAAATCAAAATGCTTTGGCTGAACAAGGTGAGGCAATTAAAGAA

GATCTTCAAGGGCTTGCAGATTTTGTAGAAGGGCAAGAGGGTAAAATTCTACAAAATGAAAC

TTCAATTAAAAAAAATACTCAGAGAAACCTTGTCAATGGGTTTGAGATTGAGAAAAATAAAG

ATGCTATTGCTAAAAACAATGAGTCTATCGAAGATCTTTATGATTTTGGTCATGAGGTTGCA

GAAAGTATAGGCGAGATACATGCTCATAATGAAGCGCAAAATGAAACTCTTAAAGGCTTGAT

AACAAACAGTATTGAGAATACTAATAATATTACCAAAAACAAAGCTGACATCCAAGCACTTG

AAAACAATGTCGTAGAAGAACTATTCAATCTAAGCGGTCGCCTAATTGATCAAAAAGCAGAT

ATTGATAATAACATCAACAATATCTATGAGCTGGCACAACAGCAAGATCAGCATAGCTCTGA

TATCAAAACACTTAAAAAAAATGTCGAAGAAGGTTTGTTGGAGCTAAGCGGTCACCTAATTG

ATCAAAAAACAGATATTGCTCAAAACCAAGCTAACATCCAAGATCTGGCCACTTACAACGAG

CTACAAGACCAGTATGCTCAAAAGCAAACCGAAGCGATTGACGCTCTAAATAAAGCAAGCTC

TGAGAATACACAAAACATCGAAGATCTGGCCGCTTACAACGAGCTACAAGATGCCTATGCCA

AACAGCAAACCGAAGCAATTGACGCTCTAAATAAAGCAAGCTCTGAGAATACACAAAACATC

GAAGATCTGGCCGCTTACAACGAGCTACAAGATGCCTATGCCAAACAGCAAACCGAAGCCAT

TGACGCTCTAAATAAAGCAAGCTCTGAGAATACACAAAACATTGCTAAAAACCAAGCGGATA

TTGCTAATAACATCAACAATATCTATGAGCTGGCACAACAGCAAGATCAGCATAGCTCTGAT

ATCAAAACCTTGGCAAAAGCAAGTGCTGCCAATACTGATCGTATTGCTAAAAACAAAGCCGA

TGCTGATGCAAGTTTTGAAACGCTCACCAAAAATCAAAATACTTTGATTGAAAAAGATAAAG

AGCATGACAAATTAATTACTGCAAACAAAACTGCGATTGATGCCAATAAAGCATCTGCGGAT

ACCAAGTTTGCAGCGACAGCAGACGCCATTACCAAAAATGGAAATGCTATCACTAAAAACGC

AAAATCTATCACTGATTTGGGCACTAAAGTGGATGGTTTTGACAGTCGTGTAACTGCATTAG

ACACCAAAGTCAATGCCTTTGATGGTCGTATCACAGCTTTAGACAGTAAAGTTGAAAACGGT

ATGGCTGCCCAAGCTGCCCTAAGTGGTCTATTCCAGCCTTATAGCGTTGGTAAGTTTAATGC

GACCGCTGCACTTGGTGGCTATGGCTCAAAATCTGCGGTTGCTATCGGTGCTGGCTATCGTG

TGAATCCAAATCTGGCGTTTAAAGCTGGTGCGGCGATTAATACCAGTGGTAATAAAAAAGGC

TCTTATAACATCGGTGTGAATTACGAGTTCTAA Protein sequence for UspA2 from ATCC strain 25238 -SEQ ID NO: 1 as described above.

Vector construction

To generate construct MC-001, the DNA fragment coding for a fragment of the UspA2 gene (amino acids 5 to 540 of ATCC strain 25238) comprising the NdeI / XhoI restriction sites to facilitate cloning (the starting methionine is encoded by the NdeI site) and the DNA sequence corresponding to the amino acid linker AS (alanine serine) and amino acids 6xhis were optimized for codons (non-native) and synthesized by GENEART (a registered trademark in the United States). -United). Optimized for codons means that the nucleotide sequence has been modified from the native sequence without changing the amino acid sequence in order to better adapt to codon usage in Escherichia coli for optimal expression. The UspA2 fragment was cloned according to standard methods into the pET-26b expression vector using the NdeI / XhoI restriction sites.

To generate constructs MC-002, MC-003, and MC-004, a polymerase chain reaction was performed to amplify the UspA2 gene fragment (amino acids 5 to 540 of ATCC strain 25238) using the construct MC-001 as template, UspA2Nde opt primer (which contains the start codon, methionine), and UspA2opt delta His primer, A2opt lHis delta AS, and A2opt 2His delta AS, respectively. The UspA2 fragment was cloned according to standard methods into the pET-26b expression vector using the NdeI / XhoI restriction sites.

To generate the MC-005 construct, a polymerase chain reaction was performed to amplify the UspA2 gene fragment (amino acids 519 to 519 of the ATCC strain 25238) using the MC-001 vector as a template with UspA2Nde primers. opt (which contains the starting codon, methionine) and delta hairpin A2opt His. The DNA sequence corresponding to the NdeI restriction site was incorporated into the 5 'primer and the XhoI restriction site was incorporated into the 3' primer. In addition, the DNA sequence corresponding to amino acid linker AS and amino acids 6xhis was incorporated into the 3 'primer. The generated PCR product was then inserted into the pET-26b (+) cloning vector (NOVAGEN (a registered trademark in the US)).

To generate construct MC-006, a polymerase chain reaction was performed to amplify the UspA2 gene fragment (amino acids 51 to 519 of ATCC strain 25238) using construct MC-005 as a template with primers UspA2Nde opt (which contains the starting codon, methionine) and delta His delta helix. The DNA sequence corresponding to the NdeI restriction site was incorporated into the 5 'primer and the XhoI restriction site was incorporated into the 3' primer. The generated PCR product was then inserted into the pET-26b (+) cloning vector (NOVAGEN (a registered trademark in the US)).

To generate construct MC-007, the DNA fragment encoding a fragment of UspA2 gene (amino acids 564 to 564 of ATCC 25238) comprising NdeI / XhoI restriction sites to facilitate cloning (the starting methionine is encoded by the NdeI site) and the DNA sequence corresponding to amino acid linker AS and amino acids 6xhis were optimized for codons and synthesized by GENEART (a registered trademark in the United States) (plasmid: 1026399) . The UspA2 fragment was cloned according to standard methods into the pET-26b expression vector using the NdeI / XhoI restriction sites.

To generate construct MC-008, a polymerase chain reaction was performed to amplify the UspA2 gene fragment (amino acids 564 to 564 of ATCC 25238) using construct MC-007 as template with primers. UspA2Nde opt (which contains the start codon, methionine) and 2His delta helix. The DNA sequence corresponding to the NdeI restriction site was incorporated into the 5 'primer and the XhoI restriction site was incorporated into the 3' primer. The generated PCR product was then inserted into the pET-26b (+) cloning vector (NOVAGEN (a registered trademark in the US)).

To generate construct MC-009, a polymerase chain reaction was performed to amplify the UspA2 gene fragment (amino acids 31 to 564 of ATCC strain 25238) using plasmid 1026399 as template and N-primers. term cyto Abis (which contains the start codon, methionine) and 2His delta helix. The DNA sequence corresponding to the NdeI restriction site was incorporated into the 5 'primer comprising the glutamine deletion and the Xhol restriction site was incorporated into the 3' primer comprising two histidine residues.

The generated PCR product was then inserted into the pET-26b (+) cloning vector (NOVAGEN (a registered trademark in the US)). DNA sequencing of the final construct was performed to confirm the correct sequence.

To generate construct MC-010, a polymerase chain reaction was performed to amplify the UspA2 gene fragment (amino acids 564 to 564 of ATCC strain 25238) using construct MC-007 as template with primers. UspA2 Nde opt (which contains the codon, starting from methionine) and cyto helix dHis dAS. The DNA sequence corresponding to the NdeI restriction site was incorporated into the 5 'primer and the XhoI restriction site was incorporated into the 3' primer. The generated PCR product was then inserted into the pET-26b (+) cloning vector (NOVAGEN (a registered trademark in the US)).

To generate construct MC-011, a polymerase chain reaction was performed to amplify the UspA2 gene fragment (amino acids 31-540 of ATCC strain 25238) using construct MC-001 as template with primers. N-termo cyto Abis (which contains the start codon, methionine) and N-term reverse. The DNA sequence corresponding to the NdeI restriction site was incorporated into the 5 'primer and the XhoI restriction site was incorporated into the 3' primer. The generated PCR product was then inserted into the pET-26b (+) cloning vector (NOVAGEN (a registered trademark in the US)).

A detailed list of PCR primer sequences used for amplifications is shown in Table 3. Polymerase chain reaction was performed using the Expand High Fidelity PCR System kit (Roche) according to the manufacturer's recommendations. Ligation was performed using the Rapid DNA Ligation kit (Roche) according to the manufacturer's recommendations.

Table 3 Sequences of PCR primers used for UspA2 amplifications

Transformation

Escherichia coli (E. coli) BLR (DE3), modified BLR (DE3) or B834 (DE3) cells were transformed with plasmid DNA according to standard methods with CaCl2-treated cells (Hanahan D. Plasmid transformation by Simanis. "In Glover, DM (Ed), DNA cloning, IRL Press London (1985): pp. 109-135.). Briefly, competent BLR (DE3) cells were thawed gently on ice. Approximately 4 μl of plasmid (10 to 100 ng) were mixed using 50 to 100 μl of competent cells. Then this formulation was incubated on ice for 5 min. To carry out the transformation reaction, the formulation was

heat pulsed at 42 ° C for 30 seconds and then incubated on ice for 2 minutes. Approximately 0.5 ml of SOC medium (Super Optimal Broth with Catabolite Suppression) was added to the transformed cells and the cell culture was incubated at 37 ° C for one hour prior to plating on Luria-Bertani (LB) agar with 50 μρ / ιηΐ of kanamycin. About 150 μl of the transformed cell culture was plated and incubated overnight at 37 ° C. BLR (DE3): BLR is a recA ~ derivative of BL21 (F-ompT hsdSB (rB- mB-) gal dem (DE3) .This strain of E. coli used for expression of recombinant proteins improves monomer yields. plasmids and can help stabilize target plasmids containing repetitive sequences or whose products may cause the loss of DE3 prophage (Studier, FW (1991) J Mol Biol 219: 37-44) The detailed genotype of E. coli BLR (DE3) was published by NOVAGEN (a registered trademark in the United States): (F-ompT hsdSB (rB- mB-) gal dem A (srl-recA) 306 :: Tn10 (TetR) (DE3 B834 (DE3) is the parental strain for BL21.These hosts are auxotrophic for methionine and allow highly specific activity for labeling target proteins with 35S-methionine and selenomethionine for crystallography. coli B834 (DE3) was published by NOVAGEN (a registered trademark in the United States): F-ompT hsdSB (rB-mB-) gal dem m and (DE3) Modified BLR (DE3): To prevent (phospho) gluconoylation, the Pgl gene was inserted into the locus of biotin located in the BLR (DE3) genome. In addition, to prevent Ile-Val substitutions, the C219Y mutation in the threonine deaminase gene has been corrected. Genotype: (F-ompT hsdSB (rB- mB-) gal dem A (srl-recA) 306 :: Tn10 (TetR); Δ (bioA-bioD) :: Pgl; TD + (C219Y) (DE3).

EXAMPLE 2 Expression of proteins using a shake flask

Escherichia coli strains transformed with a recombinant plasmid were used to inoculate 100 ml of LB broth (Becton, Dickinson and Company) ± 1% (w / v, w / v) glucose (Lab MAT, catalog number: GR-0101) and 50 μg / ml kanamycin (Sigma). This preculture was generally allowed to develop overnight at 37 ° C.

Twelve ml of preculture were used to inoculate 500 ml LB broth + 50 μg / ml kanamycin. The cultures were incubated at 37 ° C with 150 rpm shaking to reach a D.O. 600m from ~ 0, 6. At a D 0.60m of ~ 0.6, BLR cultures (DE3) were induced for expression of the recombinant protein by addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG; EMD). Chemicals Inc., Cat. No. 5815) and incubated overnight at 23 ° C with 150 rpm shaking. After the induction period, the cultures were centrifuged at 6370 g for 20 minutes and the pellets from 350 ml of culture were frozen at -20 ° C separately.

Example 3 Purification of Proteins Using Phosphate Buffer (Construction MC-001 and Construction MC-011)

Each bacterial pellet obtained after induction in a shake flask was resuspended in 30 ml of 20 mM potassium phosphate buffer (pH 8.0) containing 10 mM NaCl and Roche's COMPLETE protease inhibitor cocktail (a registered trademark in the United States) (1 tablet / 50 ml buffer). Cell lysis is performed by French 3X (20,000 psi) press extractions and clarification is by centrifugation for 30 minutes at 23,700 g. The supernatant is harvested and filtered on 0.22 μm.

6xHis-tagged proteins were purified by immobilized metal affinity chromatography (IMAC) using an XK16 column and 20 ml of NiNTA resin (Qiagen) previously equilibrated with 20 mM potassium phosphate buffer (pH 8.0) containing 10 mM NaCl or PBS buffer pH 8.0 containing 500 mM arginine. The soluble components were loaded at a rate of up to 4 ml / min (producing an "unbound fraction"). After loading onto the column, the column was washed with 60 ml of 20 mM potassium phosphate buffer (pH 8.0) containing 10 mM NaCl at a rate of 4 ml / min producing a "wash fraction no. 1 ". A second wash using the same buffer + 10 mM imidazole was performed, producing a "wash fraction no. 2 ". Elution was performed using the same buffer containing 200 and / or 500 mM imidazole.

Samples from the elution fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples containing the protein were dialyzed with 5 liters of 20 mM potassium phosphate buffer (pH 8.0) containing 10 mM NaCl. The concentration of the protein was determined using the Lowry method.

Example 4 - Protein purification using arginine-containing buffer (MC-001, MC-005 and MC-007)

Each bacterial pellet obtained after induction in a shaking flask (Example 3) or a fermenter (Example 5) was resuspended in 30 ml of PBS buffer + 500 mM arginine pH 8.0 and the cocktail of inhibitors of Roche COMPLETE proteases (a registered trademark in the United States) (1 tablet / 50 ml buffer). Alternatively, the cell fermentation paste ("7 g) was resuspended in 90 ml of PBS buffer containing 500 mM arginine pH 8.0 and Roche's COMPLETE protease inhibitor cocktail (a registered trademark at USA) (1 tablet / 50 ml buffer).

Cell lysis was performed by French 2 or 3 X (20,000 psi) press extractions and clarification was by centrifugation for 30 minutes at 23,700 g at 4 ° C. The supernatant was harvested and filtered through 0.22 μm. 6xHis-tagged proteins were purified by immobilized metal affinity chromatography (IMAC) using an XK16 column and 80 ml of NiNTA resin (Qiagen) previously equilibrated with PBS buffer + 500 mM arginine pH 8.0 . The soluble components were loaded at a rate of up to 4 ml / min (producing an "unbound fraction"). After loading on the column, the column was washed with the same buffer and then with 20 mM potassium phosphate buffer (pH 8.0) containing 10 mM NaCl at a rate of 4 to 6 ml / min producing washing fraction no. 1 ". A second wash using the same buffer + 10 mM imidazole was performed, producing a "wash fraction no. 2 ". Elution was performed using the same buffer + 200 mM imidazole or 500 mM imidazole. In other elution flasks, a final concentration of 5 mM EDTA was added.

Samples from the elution fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein-containing samples were dialyzed with 5 liters of 20 mM potassium phosphate buffer (pH 8.0) containing 10 mM NaCl and 5 mM EDTA. The concentration of the protein was determined using the Lowry method.

This protocol can be used with other proteins labeled with 6xHis.

Example 5 - Fermentation

The following fermentation procedure can be used:

The working seeds are frozen aliquots of strains derived from Escherichia coli BLR (DE3) or BLR (DE3) grown in vial, transformed with a derivative of pET26b containing a sequence coding for a recombinant protein construct candidate for a specific antigen.

Working seed (WS) is taken from the frozen storage, thawed and used to inoculate an Erlenmeyer flask containing pre-culture medium. Seed handling and vial culture are performed aseptically under a laminar airflow (FAL) host or biosafety cabinet (BSC). The vial of the preculture is incubated generally at a temperature between 30 ° C and 37 ° C under a stirring speed of 200 rpm for the time necessary to reach an optical density at 650 nm (DC> 650nm) between 1, 0 and 3.0, usually between 4 and 6 h.

A 20 l fermentor is prepared by cleaning in place followed by an automated steam sterilization sequence. The starting medium is aseptically transferred to the fermenter. A bottle filled with 25% NH40H is aseptically connected to the fermenter for automatic pH control. The initial pH of the starting medium is adjusted to the target pH by adding a solution of NH 4 OH. Antifoam is added using a syringe through a septum into the fixed plate. A bottle filled with feed medium is aseptically connected to the fermenter. The feed is controlled by either a cascade of pCu (dissolved oxygen control) or a pre-programmed feed curve. Stirring is controlled by either a cascade of pC> 2 or a pre-programmed shaking curve.

The initial parameters of the fermenter are usually as follows:

• Temperature: 28 ° C to 32 ° C • Pressure: 0.5 barg (7 psi) • Air Flow: 2 WM (filling volumes per minute) • pH: Regulated to 6.8 by adding NH4OH to 25%.

An aliquot of this preculture (usually between 5 ml and 50 ml) is used to inoculate the fermentor starting medium by addition to the syringe through a septum on the fermentor fixed plate. The phases of fermentation culture are: • Closed phase: biomass accumulates using the carbon source in the starting medium. • Powered phase: The feed medium is introduced either in a cascade control of pC> 2 or a pre-programmed feed curve. The accumulation of biomass continues on the carbon source in the feed medium. • Induction phase: the expression of the recombinant protein antigen is induced by the addition of a solution of IPTG to the culture in the fermenter. At harvest, the culture is generally collected in 1 l centrifugation bottles and centrifuged to separate the solid pellet fraction (cell paste) from the liquid supernatant fraction. The supernatant is discarded, and the wet cell weight (solid pellet) is recorded and the cell paste pockets are stored at -20 ° C.

The following procedure can also be used: Classic Escherichia coli pretreatment

Each conventional preculture was prepared using a frozen seed culture of Escherichia coli strains. These strains are BLR (DE3) strains transformed with a pET26b derivative containing a coding sequence for the specific construct to be evaluated.

The seed culture was thawed at room temperature and 400 μΐ was used to inoculate a 2 liter Erlenmeyer flask containing 400 ml of preculture medium (adapted from Zabriskie et al., J. Ind. Microbiol., 2: 87 -95 (1987)).

The inoculated flask was then incubated at 37 ° C (± 1 ° C) and 200 rpm. The preculture was stopped after 6 hours of incubation. At this step, the optical density at 650 nm (DC> 650 nm) is about 2. The preculture was used to inoculate the medium in a fermenter as soon as the culture was stopped.

Fermentation fed at 20 1 scale

Process

A 20 liter fermentor (Biolafitte) was used. Nine liters of fermentation medium in batch mode were aseptically transferred to the fermenter. The pH of the medium was readjusted to 6.8 with the addition of base. 1 ml of undiluted irradiated antifoam (SAG 471) was also added to the fermenter. The temperature (28 ° C), the head pressure (0.5 bar), the aeration rate (20 liters of air dispersed per minute) and the initial stirring speed (300 rpm) were then set before inoculation. The dissolved oxygen level under these conditions was 100%. Head pressure and aeration rate were maintained at a constant rate during fermentation. Inoculation was obtained by the addition of an equivalent of 10 ml of pre-cultured DO650nm = 2 (prepared as described above, in Example 2) according to the following formula:

During fermentation in batch mode (0 to 15 h), the temperature was maintained at 28 ° C. The dissolved oxygen level

has been set at 20%. The dissolved oxygen (DO) level was controlled by increasing agitation when the OD dropped below 20%. Glucose depletion resulted in an increase in OD and a simultaneous decrease in agitation.

When glucose is depleted, the feed rate is started based on a pH signal that rises above 7.0. From this point onwards, the feed rate was controlled by the oxygen demand, increasing the flow rate when the dissolved oxygen tended to fall below the set point at 20%. At this stage, the feeding speed is maintained at 900 rpm.

During the energized phase (before induction), the pH was maintained at 6.8 by base addition, the temperature was controlled at 30 ° C.

Two strategies have been applied to produce the protein: "High cell density induction" (HCDI) is applied when the culture is induced with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside) at an optical density of 80. ± 5, usually reached after 40 h of culture. The temperature was maintained at 28 ° C. and the feed rate was again controlled by the oxygen demand with a constant stirring speed at 900 rpm.

The "low cell density induction" (LCDI) method means an induction at an optical density of 40 ± 5 typically achieved after 24 hours of culture. The temperature was decreased to 30 ° C and the constant feed rate of 0.5 ml / min was applied. Then 1 mM IPTG is added to the culture. At this stage, the OD level was kept at 20% by controlling the stirring speed. At the end of the induction phase (72 h), the cell paste was collected by centrifugation (6500 x g, 4 ° C for 1 h), and stored at -20 ° C.

Figures 1 and 2 illustrate a typical fermentation profile with the HCDI and LCDI methods and the controlled fermenter parameters of 20 1 with a fed-batch.

Table 4 shows the constructs evaluated in the fermenter and the UspA2 yield obtained for each.

Table 4

His = histidine

Figure 3 graphically illustrates the UspA2 yield of Table 4 from the constructs evaluated in the fermenter.

In this figure, the yield of UspA2 is affected by the histidine residues present in the construction, (p <0.05, unilateral, three levels, ANOVA type II). A positive correlation has been observed between the number of histidine residues and the UspA2 yield of the fermentation, with a yield increase greater than 400% between 0 and 6 residues in the

fermentations with a semi-continuous feed ("fed-batch").

It was also observed that a histidine residue added to the half-helix motif (construct MC-003) produced a UspA2 yield of about 1 g / l of protein.

Example 6 - Protein Characterization

Analytical Ultracentrifugation Analytical ultracentrifugation is used to determine the homogeneity and size distribution of different species in a protein sample by measuring the rate at which molecules move in response to centrifugal force. This is based on the calculation of the sedimentation coefficients of the different species that are obtained by the sedimentation rate experiment, which depend on their shape and molecular mass.

The following protein samples were centrifuged in a Beckman-Coulter analytical ultracentrifuge

ProteomeLab XL-1 at 28,000 rpm after the AN-βΟΤί rotor was equilibrated at 15 ° C. at. MC-005 BMP53 lot, 675 μg / ml in 20 mM NaPC> 4.10 mM NaCl, pH 8.0b. MC-001 BMP13 lot, 545 μg / ml in 20 mM NaPC> 4.10 mM NaCl, pH 8.0 c. MC-001 BMP14 lot, 545 μg / ml in 20 mM NaPO 4, 10 mM NaCl, pH 8.0 d. MC-001 BMP54 lot, 445 μg / ml in 20 mM NaPO 4, 10 mM NaCl, pH 8.0 e. MC-007 BMP70 lot, 510 μg / ml in 20 mM NaPO 4, 10 mM NaCl, pH 8.0

For data collection, 133 to 325 readings were recorded at 280 nm every 5 minutes. Data analysis was performed using the SEDFIT program (available through the National Institutes for Health) for determining the distribution of C (S). The distribution of C (S) is a representation of the relative intensity of the different components in a mixture of macromolecules separated by their sedimentation coefficient, which is a function of the size and conformation of the molecules. Determination of the partial specific volume of the proteins at 15 ° C was performed with the SEDNTERP software from their amino acid sequence. SEDNTERP (SEDNTERP is distributed and supported by the Biomolecular Interaction Technologies Center at the University of New Hampshire) was also used to determine the viscosity and density of the buffer at 15 ° C.

The determination of the relative abundance of all species was made by considering the total area under the curve of the global distribution as 100% of the sample and calculating the percentage of this total area represented by the contribution of each species. The plot of the C (S) distribution (concentration versus sedimentation coefficient) was used for this calculation, considering that it is a better representation of the raw data than the distribution of C (M) (concentration according to the molecular weight). Analytical ultracentrifugation of the different purified constructs allowed observation that UspA2 Ahelice, UspA2 ½ helix and UspA2 full helix with a C-terminal his tag are present mainly in the form of trimers in solution when 500 mM L-arginine is added during cell lysis prior to purification (Figures 4, 5, 7 and 8).

A heterogeneous size distribution for UspA2 ½ helix was observed when no L-arginine was added during cell lysis. Two major populations are observed. It was not possible to confirm the molecular weight of the species detected by AUC (analytical ultracentrifugation) with this protein preparation, because the friction ratio that is essential for molecular weight estimation must be calculated from of a homogeneous sample. However, based on the sedimentation coefficients, none of the species observed appears to correspond to the trimer observed in other samples.

Figure 4 illustrates the molecular weight distribution of purified MC-005 determined by the rate of sedimentation by analytical ultracentrifugation. The majority of the protein is found as a trimer, with a small proportion of a higher molecular weight oligomer that may be a dimer of trimers.

Figure 5 illustrates the molecular weight distribution of purified MC-001 determined by the rate of sedimentation by analytical ultracentrifugation. The majority of the protein is found in trimer form.

Figure 6 illustrates the molecular weight distribution of purified MC-001 determined by the rate of sedimentation by analytical ultracentrifugation. The sample has multiple species and is highly polydispersed. The sedimentation coefficient of the main species detected does not correspond to one of the trimers normally detected in the other batches.

Figure 7 illustrates the molecular weight distribution of purified MC-001 determined by the rate of sedimentation by analytical ultracentrifugation. The majority of the protein is found in trimer form.

Figure 8 illustrates the molecular weight distribution of purified MC-007 determined by the rate of sedimentation by analytical ultracentrifugation. The majority of the protein is found in trimer form.

Circular Dichroism / Secondary Structure

Circular dichroism (CD) is used to determine the composition of secondary structures of a protein by measuring the difference in absorption of left-polarized light versus right-polarized light that is due to structural asymmetry. The shape and amplitude of the CD spectra in the far UV region (190 to 250 nm) are different if a protein exhibits a beta sheet, an alpha helix, or a random coil structure. The relative abundance of each type of secondary structure in a given protein sample can be calculated by comparison with the reference spectra.

Far-UV spectra are measured using a 0.01 cm optical path from 178 to 250 nm, with a resolution of 1 nm and a bandwidth on a Jasco J-720 spectropolarimeter. The temperature of the cell is maintained at different temperatures by a Peltier thermostated cell block RTE-111. A nitrogen current of 10 1 / min is maintained during the measurements.

The concentration of the following protein constructs was adjusted to 400 μg / ml in a buffer of 20 mM NaPO 4, 10 mM NaCl, pH 8.0. at. MC-005 BMP53 batch, in 20mM NaPO4, 10mM NaCl, pH 8.0b. MC-001 BMP13 batch, in 20 mM NaPO 4, 10 mM NaCl, pH 8.0 c. MC-001 BMP14 batch, in 20 mM NaPO 4, 10 mM NaCl, pH 8.0 d. MC-001 lot BMP54, in 20 mM NaPO4, 10 mM NaCl, pH 8.0 e. MC-007 BMP70 batch, in 20 mM NaPO 4, 10 mM NaCl, pH 8.0

Secondary structure calculations were performed using the following algorithms:

Selcon 3 (Sreerama and Woody, Anal Biochem (1993), 209, 32, Sreerama and Woody, Biochemistry, 33, 10022-25 (1994);

Sreerama et al. Protein Science, 8, 370-380 (1999); Johnson W.C. Jr., Proteins: Str. Func. Broom. 35, 307-312 (1999)) CDSSTR (Johnson W.C. Proteins: Struc Func Genet 35, 307-312 (1999) modified by Sreerama, N. (Anal Biochem, 287, 252 (2000)).

The displayed results are an average of the percentage calculated with the two algorithms and they are subject to a margin of error of 5%.

The results of the secondary structure calculations for the proteins expressed in the fermenter are presented in Table 5, taking into account a margin of error of R s.

Table 5

Secondary structure calculations at 22 ° C

The calculations are consistent with the shape and visual analysis of the spectra, where the helix content increases with the intensity of the minima at 208 and 220 nm. Proteins are

composed of a high proportion of helical structures, with the presence of beta structures.

The superposition of the spectra in FIG. 9 shows no significant difference in shape between the constructions. The MC-005 helix spectrum shows a lower intensity that could be responsible for a lower alpha structure that is consistent with the absence of C-terminal helix.

Figure 9 illustrates the far UV circular dichroism spectrum of the UspA2 constructs MC-001, MC-005 and MC007 giving an indication of the secondary structures of the proteins. The superposition of the spectra clearly shows that the constructions containing a half-helix or a C-terminal whole helix show no detectable difference in their secondary structures, whereas the helical construction generates a spectrum with a difference in intensity that could be responsible. of different content of secondary structure. Thermal unfolding

Measuring CD spectra in far UV at different temperatures during thermal unfolding suggested that MC-005 is less heat-stable than MC-007. The spectrum observed at 33 ° C for MC-005 is similar to the standard spectrum of unfolded protein. For MC-007 construction, although partial loss of secondary structures is observed at 33 ° C, complete unfolding appears to occur between 35 ° C and 37 ° C. This may be an indication of superior thermal stability of the MC-007 construction containing an entire helix.

Figure 10 illustrates secondary structure tracking by circular dichroism during thermal unfolding of MC-005 (UspA2Aelice + 6His). Visual analysis of the spectrum clearly shows that the protein loses most of its secondary structures at 33 ° C.

Figure 11 illustrates the secondary structure tracking by circular dichroism during thermal unfolding of MC-007 (UspA2 + helix + 6His). Visual spectrum analysis shows that the loss of secondary structure is slower compared to the propeller-free construction. Structural changes are detectable upon heating at 33 ° C, but full unfolding appears to occur between 35 ° C and 37 ° C. Thermal Unfolding by Differential Scanning Calorimetry (DSC)

Thermal transitions of different UspA2 constructs were compared to evaluate the effect of C-terminal helical changes on the thermal stability of proteins. The analysis was performed on VP-DSC from MicroCal (part of GE Healthcare). The buffer of 20 mM NaPO4, 10 mM NaCl, 5 mM EDTA, pH 8 was used as a reference and subtracted from the readings. The proteins were equilibrated at the initial temperature for 15 minutes before the temperature rise. The DSC readings were then conducted from 10 ° C to 60 ° C at a heating rate of 90 ° C / hr.

Two transitions were detected in constructs MC-001 and MC-007 and only one in MC-005. The values of the transitions (or Tm) of the different constructions can be found in Table 6.

While the lower Tm of the three proteins is around 32 ° C, the main difference is the value of the second Tm. The whole helix-containing construct (MC-007) has a Tm greater than 37.5 ° C compared to 34.5 ° C for the half-helix (MC-001).

It has been shown that for MC-001 and MC-007, the first Tm around 32 ° C is reversible, while the higher Tm is irreversible. For MC-005, the only Tm detected is irreversible.

This may be an indication of superior thermal stability of the MC-007 construction containing an entire helix.

Table 6

Fusion points of UspA2 constructs measured by DSC

Mass spectrometry

UspA2 protein samples were prepared by protein precipitation with the CHCI3 / MeOH / H2O system. The pellet of the proteins was centrifuged at the bottom of the Eppendorf tube before being gently dried under nitrogen. The dried pellet was then dissolved in 2 μl of pure formic acid before being diluted with 3 μl of ultrapure water and 5 μl of sinapinic acid. The sinapinic acid used as a template for the MALDI-TOF (Matrix Assisted Laser Desorption and Ionization Time-of-Flight Spectrometry Analyzer) is prepared in 50% CH3CN / 50% H20 supplemented with TFA at a final concentration of 0.1%. 1 μl of the sample + matrix mixture was deposited on a MALDI Bruker 384 stainless steel target rectified and allowed to dry for crystallization at room temperature and atmospheric pressure (dried droplet method). Mass spectrometry analysis of UspA2 was performed on a Bruker Ultraflex 2 MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in positive and linear ionization mode. Protein samples, co-crystallized in the sinapinic acid matrix, were irradiated with a SmartBeam laser. The mass measurement of the intact UspA2 protein was performed over a mass range of 10,000 to 100,000 Da with

an acceleration voltage of 25 kVolts. The attenuation of the laser has been refined in order to obtain the best possible protein signal and to avoid any fragmentation as well as the phenomenon of over-ionization of the background noise. Calibration of the mass spectrometer was performed in a close external procedure with a homologous template and using the commercial protein Bruker Protein Calibration mixture 2, by exact measurements of the following standards: [M + 2H] 2+ (mass measured by an MS detector after the addition of two H + ions to the protein during ionization) species of protein A at m / z 22307 Da, [M + H] + trypsinogen species at m / z 23982 Da, [ M + H] + species of protein A at m / z 44613 Da and [M + H] + bovine albumin species at m / z 66431 Da. Each spectrum presented comes from the sum of 500 individual injections.

The following samples were analyzed:

Construction MC-001 with amino acids MQAK (SEQ ID NO: 85) in N-terminal produced in stirred flask, Opt-01 batch, MC-011 construction with amino acids MAK in N-terminal produced in stirred flask, lot BMP37.

In Table 7 and Figure 12, it has been shown that the MC-001 protein with amino acids MQAK (SEQ ID NO: 85) at the N-terminus is at least partially demethionylated, as shown by the measured molecular mass of 57427 Da, compared to the expected mass of 57565 Da. The other peak of 57620 Da may represent the complete non-demethionylated protein, N-acetylated protein, or other population of the modified protein.

Figure 12 illustrates the MALDI spectrum of MC-001 lot opt-01. The mass observed at 57427 Da may be consistent with the demethionylated protein, while the peak at 57620 Da could correspond to the complete protein.

As shown in Table 7 and Figure 13, the MC-011 protein with MAK amino acids at the N-terminus resulted in a major population of MALDI-MS which may correspond to the demethionylated protein, with mass of 572 65 Da, compared to 57437 Da for the expected mass based on the complete amino acid sequence. The other two peaks at +186 Da and +366 Da are not close to any expected posttranslational changes, so they could not be identified by this experiment.

Table 7

Molecular weight of two UspA2 constructs measured by MALDI-TOF. Both constructs have a lower main measured mass than expected from the amino acid sequence. The mass of the major population obtained with the two constructs can be consistent with a demethionylated protein

N-terminal sequencing by Edman degradation

In order to evaluate whether optimization of the N-terminal region (optimization of the amino acid sequence next to the N-terminal methionine) leads to demethionylation of the protein, N-terminal sequencing was performed on the MC-011 construct carrying amino acids MAK on its N-terminus.

Proteins were separated by SDS-PAGE on a Novex polyacrylamide gel from 4% to 20% Invitrogen, before transfer to PVDF membrane Problem (polyvinylidene difluoride) (Bio-Rad). The membrane was stained by Amidoblack. The band of interest was then cut out and the analysis was performed according to the manufacturer's protocol using an Applied Biosystems Procise sequencing system. Twelve Edman degradation cycles were performed.

The N-terminal amino acid sequence obtained is AKNDITLEDLP (SEQ ID NO: 86), which corresponds to the N-terminus of the protein starting at amino acid number two after the initial methionine. This indicates that the mature protein is predominantly demethionylated.

Example 7 - Construction of UspA2 MC-001: bactericidal activity Bactericidal test

Moraxella catarrhalis was grown overnight on a Petri dish at 37 ° C + 5% CO2. Bacteria were transferred into 12 ml of 0.1% HBSS-BSA buffer (Hank's buffered saline with bovine serum albumin) to obtain an OD 600 of 0.650. The serum samples were heated for 45 minutes. at 56 ° C to inactivate the endogenous complement. Half-serial dilutions of the sera in SBA buffer (HBSS-BSA 0.1%) were added on a 96-well microtiter plate with a rounded bottom (25 μl / well). Then 50 μl of SBA buffer was added to each well. Then 25 μl of Moraxella catarrhalis strains at 4 × 10 4 cfu / ml were added to the wells containing the sera and incubated for 15 min at room temperature Finally, 25 μl of freshly thawed baby rabbit supplement diluted 1/8 in HBSS-BSA 0.1% was added to reach a final volume of 125 μl The plates were incubated for 1 hour at 37 ° C. with orbital shaking (210 rpm). depositing the microplate on ice for at least 5 min.

After homogenization, various dilutions of the suspension (a mixture of bacteria, serum, complement and buffer, at a volume of 125 μΐ as described in the previous paragraph) were added to chocolate agar plates and incubated. for 24 hours at 37 ° C with 5% CO2 and colonies of Moraxella catarrhalis were counted.

Eight wells without a serum sample were used as bacterial controls to determine the number of Moraxella catarrhalis colonies per well. The average number of CFUs (colony forming unit) of the control wells was determined and used to calculate the destructive activity for each serum sample. The bactericidal titres were expressed at the reciprocal dilution of serum inducing 50% of destruction.

Anti-UspA2 antisera generated in mice, guinea pigs and rabbits against MC-001 were tested in the bactericidal test described hereinabove against 20 different strains of Moraxella catarrhalis isolated from various tissues (blood, sputum, nose, fluids). middle ear) in various countries (USA, Finland, Netherlands, Norway, Sweden).

As shown below, anti-UspA2 antibodies were able to induce cross-bacterial destruction of Moraxella catarrhalis, regardless of the percentage of UspA2 homology expressed by the strain tested. In addition, bactericidal activity was also shown against strains that express only UspA1 or UspA2H chimeric protein. As expected, no bactericidal antibody titer or only low bactericidal antibody titers were measured against double knockout mutants for UspA1 and UspA2.

Table 8

Cross-bactericidal activity of UspA2 MC-001 antibodies generated in mice, guinea pigs and rabbits. 1 + 2 KO means double knockout, for UspA and UspA2. 1KO represents knockout only for UspAl. MEF (AOM) = fluid of the middle ear (acute otitis media). "/" In the source column of the isolate = source of the unknown isolate

Table 9

Expression of UspA in M. catarrhalis strains in Table 8

Example 8 - Protection in a Mouse Model of Colonization of the Lung (MC-001)

Female five week old Balb / c mice (n = 8/5 groups) were immunized by the intramuscular route on days 0, 14 and 28 with 50 μl of vaccine containing 10 μg UspA2 MC-001 formulation formulated within of AS02V. The mice were challenged intranasally at day 42 with 5 × 10 CFU of various strains of Moraxella catarrhalis. Bacteria were counted in the lungs taken at 0, 3 and 6 hours post challenge. The differences between the groups were analyzed using the Dunnet test.

As summarized in Table 10, the construction of UspA2 MC-001 induced significant protection against both homologous and heterologous strains, including the 43617 strain that expresses UspA1 but not UspA2, and the BBH18 strain that expresses the protein. chimeric UspA2H (consisting of the N-terminal sequence of UspA1 and the C-terminal sequence of UspA2).

Table 10

Protective efficacy of UspA2 MC-001 construction

determined using the GapL / ClustaIX software against the UspA2 ATCC25238 AA fragment at 540. the p values in bold are significant (p <0.05)

Example 9 - Construction of UspA2 MC-007: Bactericidal Activity of Antibodies

Bactericidal test

Moraxella catarrhalis 25238 was grown overnight on a Petri dish at 37 ° C + 5% CO2. Bacteria were transferred into 12 ml of 0.1% HBSS-BSA buffer to obtain an OD600 of 0.650. The serum samples were heated for 45 min at 56 ° C to inactivate the endogenous complement. Half-serial dilutions of the sera in SBA buffer (HBSS-BSA 0.1%) were added on a 96-well microtiter plate with a rounded bottom (25 μl / well). Then 50 μl of SBA buffer was added to each well. Then 25 μl of Moraxella catarrhalis strain 25238 at 4 × 10 4 cfu / ml were added to the wells containing the sera and incubated for 15 min at room temperature Finally, 25 μl of freshly thawed baby rabbit supplement diluted 1/8 in 0.1% HBSS-BSA was added to reach a final volume of 125 μl The plates were incubated for 1 hour at 37 ° C. with orbital shaking (210 rpm) The reaction was stopped depositing the microplate on ice for at least 5 min After homogenization, various dilutions of the suspension were added to chocolate agar plates and incubated for 24 hours at 37 ° C with 5% CO2 and the colonies eight wells without a serum sample were used as bacterial controls to determine the number of Moraxella catarrhalis colonies per well, and the average number of CFUs in the control wells was determined. and used to calculate the destructive activity for each serum sample. The bactericidal titres were expressed at the reciprocal dilution of serum inducing 50% of destruction.

UspA2 antisera generated in mice with the construction of UspA2 MC-001 or MC-007 were tested in the bactericidal assay described above against the homologous strain of Moraxella catarrhalis 25238.

As shown in Table 11, the construction of UspA2 MC-007 elicited a high bactericidal response, similar to that induced by MC-001.

Table 11

Bactericidal activity of UspA2 antibodies MC-001 and MC-007. Normal mouse sera = mouse sera immunized with AS02V only, not with UspA2

Example 10 - Construction of UspA2 MC-007: Protective Efficacy in a Lung Test Model

Protection in a mouse model of colonization of the lung Five week old female Balb / c mice (8 mice per group, 5 groups maximum per time point) were immunized by the intramuscular route on days 0, 14 and 28 with 50 μΐ of vaccine containing 10 μg of UspA2 construct MC-001 formulated with AS02V or MC-007 formulated within AS02V. The mice were challenged intranasally at day 42 with 5 × 10 5 CFU of Moraxella catarrhalis strain ATCC (US registered trademark) 25238 ™. The mice were immunized with 10 μg of killed whole cells of Moraxella catarrhalis strain ATCC (a registered trademark in the United States) 25238 ™ (as a positive control) (WC Cat.

in Figure 14) or with AS02V alone (as a negative control). Bacteria were counted in the lungs taken at 0, 3 and 6 hours post challenge. The differences between the groups were analyzed using the Dunnet test.

As shown in Figure 14, the two UspA2 constructs were similarly protective against the ATCC (US registered trademark) strain 25238 ™.

Example 11 Immunogenicity of UspA2 MC-009 Protein Formulations in Mice

Groups of 25 female Balb / c mice were immunized by the intramuscular (IM) route at days 0, 14 and 28 with 50 μl of the following formulations: -MC-009 (1 μg) A1P04 (1000 μg / ml) -MC -009 (1 μg) AS04C (A1PO4 / MPL 100/100 per ml) -MC-009 (1 μg) AS01E (QS21 / MPL 50/50 per ml)

Anti-IgG levels were determined in the individual sera taken at day 28 (PII) and 42 (PIII) using the following protocol:

ELISA test to measure antibodies against UspA2

Plates were overlaid overnight at 4 ° C with 100 μl per well of UspA2 MC-009 at 4 μg / ml in pH 9.6 carbonate buffer. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2O2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well (100 μl). / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter).

Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

As shown in Figure 15, UspA2 induced elevated antibody levels with each adjuvant formulation.

Bactericidal test

The bactericidal assay was performed against the strain of M. catarrhalis ATCC (a registered trademark in the United States) 25238 ™ expressing a full-length homologous UspA2 using the following protocol: the strain of Moraxella catarrhalis ATCC (a registered trademark in the United States). United) 25238 ™ was grown overnight on a Petri dish at 37 ° C + 5% CO2. Bacteria were transferred into 10 ml of BHi (heart infusion broth) to obtain an OD 650 of 0.650. The serum samples were heated for 45 min at 56 ° C to inactivate the endogenous complement. Half-serial dilutions of the sera in SBA buffer (HBSS-BSA 0.1%) were added on a 96-well microtiter plate with a rounded bottom (25 μl / well). Then 50 μl of SBA buffer was added to each well. Then 25 μl of Moraxella catarrhalis 25238 ™ strains at 4 × 10 -3 cfu / ml were added to the wells containing the sera and incubated for 15 min at room temperature Finally, 25 μl of freshly thawed baby rabbit supplement diluted to 1 / In 0.1% HBSS-BSA, the plates were incubated for 1 h at 37 ° C with orbital shaking (210 rpm). stopped by placing the plate on ice for at least 5 minutes, then a 20 μl aliquot of each well of the plate was transferred to the corresponding well of a 96-well round bottom microplate and 50 μl of 0.9% Mueller Hinton broth was added to each well, 50 μl 0.9% PBS was added as a second layer, and after 3 hours at 37 ° C with 5% CO2, plates were added. incubated overnight at 25 ° C, the colonies of Morax ella were enumerated using an automated image analysis system (KS 400, Zeiss, Oberkochen, Germany). Eight wells without serum samples were used as bacterial controls to determine the number of Moraxella per well. The average number of CFUs of the control wells was determined and used to calculate the destructive activity for each serum sample. The bactericidal titres were expressed at the reciprocal dilution of serum inducing 50% of destruction.

Figure 16 illustrates the bactericidal titres induced by UspA2 against a homologous strain. In this experiment, UspA2 induced high levels of bactericidal antibodies for each adjuvant formulation. The sera were tested at PIII; five groups of five sera samples were tested.

Example 12 - Immunogenicity of UspA2 in Combination with NTHi PD and PE-PilA Antigens

Immunization protocols

Groups of 25 female Balb / c mice were immunized by the intramuscular (IM) route on days 0, 14 and 28 with 50 μl of the following formulations: - UspA2 construct MC-009 (1 μg) A1P04

- construction of UspA2 MC-009 (1 pg) AS04C

- construction of UspA2 MC-009 (1 pg) AS01E

UspA2-PD-PEPilA (construction of UspA2 MC-009, construction of PEPilA LVL-735) A1P04 (1 μg each of UspA2, PD and PEPilA, 1000 mg / ml AlPO4)

UspA2-PD-PEPilA (construction of UspA2 MC-009, construction of PEPilA LVL-735) AS04C A1P04 (1 μg of each of UspA2, PD and PEPilA, 100/100 per ml of AlPO4 / MPL)

UspA2-PD-PEPilA (construction of UspA2 MC-009, construction of PEPilA LVL-735) AS01E (1 μg of each of UspA2, PD and PEPilA, 50/50 per ml of QS21 / MPL)

ELISA test to measure antibodies against UspA2

Anti-UspA2 IgG levels were determined in the individual sera taken at days 28 and 42 using the following protocol.

Plates were overlaid overnight at 4 ° C with 100 μl per well of UspA2 MC-009 at 4 μg / ml in pH 9.6 carbonate buffer. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2C> 2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well ( 100 μΐ / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter). Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

ELISA test to measure anti-PE antibodies

Plates were overlaid overnight at 4 ° C with 100 μΐ per well of 2 μρ / μηΐ UspA2 in pH 9.6 carbonate buffer. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2O2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well (100 μl). / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter).

Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

ELISA test to measure anti-PilA antibodies

Plates were coated overnight at 4 ° C with 100 μΐ per well of 4 μρ / ιηΐ PilA in carbonate buffer pH 9.6. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2O2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well (100 μl). / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter).

Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

ELISA test to measure anti-PD antibodies

Plates were overlaid overnight at 4 ° C with 100 μΐ per well of 8 μρ / ιηΐ PD in carbonate buffer pH 9.6. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2O2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well (100 μl). / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter).

Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

Bactericidal test

Bactericidal titres were measured in pooled sera (5 groups of serum / group) taken at day 42 using the following protocol:

Moraxella catarrhalis was grown overnight on a Petri dish at 37 ° C + 5% CO2. Bacteria were transferred into 10 ml of BHi (heart infusion broth) to obtain an OD 650 of 0.650. The serum samples were heated for 45 min at 56 ° C to inactivate the endogenous complement. Half-serial dilutions of the sera in SBA buffer (HBSS-BSA 0.1%) were added on a 96-well microtiter plate with a rounded bottom (25 μl / well). Then 50 μl of SBA buffer was added to each well. Then 25 μl of strains of Moraxella catarrhalis 25238 at 4 × 10 -3 cfu / ml were added to the wells containing the sera and incubated for 15 min at room temperature Finally, 25 μl of freshly thawed baby rabbit supplement diluted 1/8 in 0.1% HBSS-BSA was added to reach a final volume of 125 μl The plates were incubated for 1 hour at 37 ° C. with orbital shaking (210 rpm) The reaction was stopped depositing the plate on ice for at least 5 minutes A 20 μl aliquot of each well of the plate was then transferred to the corresponding well of a 96-well round bottom microplate and 50 μl of 0-well agar plate. 9 ml of Mueller Hinton broth was added to each well, 50 μl of 0.9% PBS agar was added as the second layer, and after incubation for 3 hours at 37 ° C with 5% CO2, the plates were incubated. overnight at 25 ° C. Moraxella colonies were enumerated using an automated image analysis system (KS 400, Zeiss, Oberkochen, Germany). Eight wells without serum samples were used as bacterial controls to determine the number of Moraxella per well. The average number of CFUs of the control wells was determined and used to calculate the destructive activity for each serum sample. The bactericidal titres were expressed at the reciprocal dilution of serum inducing 50% of destruction.

The bactericidal test was performed against the Moraxella catarrhalis strain 25238 ™, expressing a homologous UspA2.

A negative impact of the presence of PD and PE-PilA antigens on anti-UspA2 IgG levels in AS04C (post III) and AS01E (post II) formulations was observed (Figure 17). However, the impact remained limited (2 fold of antibody decrease) and was not confirmed in the bactericidal test (Figure 18). Induced IgG responses against PD, PE and PilA antigens in mice by PE-PEPilA-UspA2 vaccine are shown in Figure 19, Figure 20 and Figure 21, respectively.

Therefore, UspA2 was immunogenic when combined with PD and PE-PilA antigens.

Example 13 - Construction of UspA2 MC-009: Immunogenicity of PD and PE-PilA antigens of NTHi in combination with UspA2 in mice

Immunization protocol

Groups of 25 female Balb / c mice were immunized by the intramuscular (IM) route at days 0, 14 and 28 with 50 μl of the following formulations:

- PD-PEPilA (1 μρ of PD and 1 μρ of construction of PEPilA LVL-7 35) AS OIE

- UspA2-PD-PEPilA (1yg UspA2 MC-009 construction, PD and PEPilA LVL-735 construction) AS01E

IgG levels by ELISA against PD, PE and PilA antigens were determined in individual sera taken at days 28 (PII) and 42 (PIII).

ELISA test to measure anti-PE antibodies

Plates were overlaid overnight at 4 ° C with 100 μΐ per well of 2 μρ / ml UspA2 in pH 9.6 carbonate buffer. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2O2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well (100 μl). / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter).

Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

ELISA test to measure anti-PilA antibodies

Plates were coated overnight at 4 ° C with 100 μΐ per well of 4 μρ / ιηΐ PilA in carbonate buffer pH 9.6. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2C> 2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well ( 100 μΐ / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter).

Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

ELISA test to measure anti-PD antibodies

Plates were overlaid overnight at 4 ° C with 100 μΐ per well of 8 μρ / ιηΐ PD in carbonate buffer pH 9.6. The plates were washed three times with 0.09% NaCl, TWEEN (a registered trademark in the United States) (0.05% polysorbate 20). After washing, half-serial dilutions of the sera were added to the microwells in PBS, 0.05% TWEEN (a registered trademark in the US). The plates were placed at room temperature for 30 minutes with stirring. After washing, mouse anti-mouse IgG antibodies (Jackson 115-035-003) conjugated to peroxidase (100 μl per well) were added, and the plates were placed at room temperature for 30 minutes with shaking. The plates were washed as above and the revealing solution (4 mg of OPDA Sigma P8787 and 5 μl of H2O2 in 10 ml of 0.1 M citrate pH 4.5) was added to each well (100 μl). / well) for 15 min in the dark. The reaction was stopped by the addition of 50 μl 1N HCl and the absorbance was read at 490 nm (620 nm for the reference filter).

Titles were calculated using the 4-parameter method using SOFTMAX software (a registered trademark in the United States) Pro.

No major impact of the addition of UspA2 on the immunogenicity of PD and PEPilA in AS01E was observed as shown in Figures 22, 23 and 24.

Example 14 - Safety of a tetravalent vaccine formulation containing UspA2 in a mouse model of lung inflammation caused by Moraxella catarrhalis

To mitigate the risk of inducing unwanted inflammatory responses in the lungs of COPD patients after immunization with a candidate vaccine to prevent exacerbations due to nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (M. cat.), Various Animal models have been developed and used to estimate the safety of this vaccine. The test formulation contained three NTHi antigens (PD, PE and PilA, with the latter two combined as a PEPilA fusion protein), a M. cat antigen. (UspA2) and the adjuvant system 01E (ASOIe).

Two models were particularly dedicated to evaluate the safety of the UspA2 component of the vaccine.

Model 1: • Objective

This model aimed to estimate the possible induction of undesirable immune responses in inflamed lungs after vaccination. • Study design

C57B1 / 6 mice were sensitized by three intranasal administrations of 25 μg strain of M. cat. ATCC (a registered US trademark) 25238 ™ heat inactivated whole cell (expressing a UspA2 that is 100% homologous to the UspA2 vaccine) on days 0, 7 and 14. This treatment induced in the lungs perivascular and peribronchiolar inflammation (with formation of lymphoid aggregates), alveolitis, pneumonia, fibrosis and a strong response of M. cat-specific IL-17 + CD4 + T cells. whole cell, which overall mimicked the inflammatory process observed in the lungs of COPD patients (with the exception of emphysema).

The mice were then vaccinated at day 42 by the intramuscular route with 1/10 of the human dose of the following formulations: PD 10 μg / PEPilA (construction LVL735, described in document WO 2012/139225) 10 μg / UspA2 (construction MC009) 10 μg / ASOIe-PD 10 μg / PEPilA (LVL735 construct) 10 μg / UspA 2

(construction MC009) 3.3 μg / AS01E - ASOIe (negative control) - PBS (negative control)

To estimate the impact of these formulations on the inflammation of the lung induced by sensitization: The mice were followed daily from day 43 to day 49 to observe mortality and any clinical sign indicating the induction of adverse events (prostration , piloerection, vaulted position). A histological analysis of the lungs was performed on days 2, 7 and 14 after vaccination (with 5 mice per group and time point) to observe a possible aggravation of inflammation. Induction of potentially unwanted T-cell responses was assessed in groups of lungs collected at days 7 and 14 post-vaccination (with 4 groups of lungs / group / time point and lungs of 3 mice per group of lungs). ). T cells of the lungs were restimulated overnight with either UspA2, M. cat. heat-inactivated whole cells (WC) of either medium (as a negative control) and then analyzed by flow cytometry for CD5, CD4, CD8, IL-17, IL-13, TNF? and IFN? expression. • Results

No deaths or adverse events were reported. - Lung Histology (Figures 25 to 29): o Changes observed in the lungs were similar in severity in all groups and characterized by mild to moderate perivascular / bronchiolar mononuclear cell infiltrates. No alveolitis and / or pneumonia associated with vaccination was observed. - T cell response: Strong CD4 + T cell responses (mainly IL-17 and TNFa-producing cells) were measured in the lungs after restimulation with WC, but without taking into account the formulation administered (vaccines or adjuvant alone or PBS) (Figures 30-33). Low or no CD8 + T cell responses were observed in the lungs (data not shown). o No detectable T-cell responses were restimulated by UspA2 peptides, regardless of group, indicating that no specific UspA2 response was initiated or stimulated after vaccination (data not shown).

Model 2: • Objective

This model aimed to estimate the possible induction of undesirable immune responses in inflammed lungs after vaccination and challenge with M. cat. • Study design

C57B1 / 6 mice were successively: Sensitized by three intranasal administrations of 25 μg of the strain of M. cat. 25238 to heat-inactivated WC (expressing a UspA2 that is 100% homologous with UspA2 vaccine) at days 0, 7 and 14 (as in Model 1). - Vaccinated at day 42 by the intramuscular route with 1 / 10th of the human dose of the following formulations (as in model 1):

PD (10 μg / PEPilA (LVL735 construct) 10 μg / UspA2 (MC009 construct) 10 μg / AS01E PD 10 μg / PEPilA (LVL735 construct) 10 μg / UspA 2

(MC009 construct) 3.3 μg / AS01E ASOIe (negative control) PBS (negative control) - Tested by intranasal administration of 25 μg of the M. cat strain. Fl0 to heat-inactivated WC (expressing a UspA2 that shares 53% homology with UspA2 vaccine) or by intranasal administration of PBS as a control, both at day 56. The strain of challenge was different from the sensitization strain to mimic the situation observed in patients with COPD who are experiencing new exacerbations due to M. cat strains. newly acquired.

To estimate the impact of vaccination and challenge on lung inflammation induced by sensitization: Mice were followed daily from day 43 to day 63 to observe mortality and any clinical signs indicative of induction of adverse events (prostration, piloerection, vaulted position). Induction of potentially unwanted T cell responses was assessed on groups of lungs taken at days 7 and 14 post challenge (with 4 groups of lungs / group / time point and lungs of 3 mice per group of lungs). T cells of the lungs were restimulated overnight with either UspA2, M. cat. F10 to WC inactivated by either medium heat (as a negative control) and then analyzed by flow cytometry for expression of CD5, CD4, CD8, IL-17, IL-13, TNFα and IFNγ. • Results - No mortality or adverse events were reported. - T cell responses: o Strong CD4 + T cell responses after challenge (mainly cells producing IL-17 and TNFa) were measured in the lungs after F10-WC restimulation, regardless of the administered formulation (vaccines or adjuvant alone or PBS) (Figures 34 to 37). Not surprisingly, these responses were higher in mice challenged with inactivated bacteria compared to mice challenged with PBS. Regardless of the challenge, low or no CD8 + T cell responses were observed (data not shown). o No detectable T cell responses were restimulated by UspA2 peptides, regardless of group, indicating that no specific UspA2 response was initiated or stimulated after challenge (data not shown).

Conclusion

It has been shown that the tested PD / PEPilA / UspA2 / ASOIe formulations and more specifically the UspA2 component of these vaccines were safe in a M. cat-induced lung inflammation mouse model.

Claims (40)

1. Protein of formula I: wherein: A represents UspA2 of Moraxella catarrhalis or an immunogenic fragment thereof; Ri represents an amino acid; m is 0 or 2; B represents a histidine; and n is 0, 1, 2, 3, 4, 5 or 6. 2. Protein according to claim 1, wherein A represents an immunogenic fragment of UspA2. 3. Protein according to any one of claims 1 and 2, wherein A represents UspA2 of any one of SEQ ID NO: SEQ ID NO: 38 therein. 4. Protein according to any one of claims 1 to 3, wherein m is 2. 5. Protein according to any one of claims 1 to 4, wherein m is 0. 6. Protein according to any one of claims 1 to 5, wherein (Ri) m represents AS (alanine serine). A protein according to any one of claims 1 to 6, wherein n is selected from the group consisting of 1, 2 and 6. Protein according to any one of claims 1 to 7, wherein n is 2. The protein of any one of claims 1 to 8, further comprising a methionine at the amino terminus. 10. Protein according to any one of claims 1 to 9, wherein n is 0. A protein according to any one of claims 1 to 10, wherein A is UspA2, where UspA2 is at least 63%, 66%, 70%, 72%, 74%, 75%, 77%, 80% identical. , 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, over the entire length, with SEQ ID NO: 1. A protein according to any one of claims 1 to 10, wherein A is an immunogenic fragment of UspA2 selected from the group consisting of amino acids 30 to 540 of SEQ ID NO: 1 (SEQ ID NO: 39), the acids Amines 31 to 540 of SEQ ID NO: 1 (SEQ ID NO: 40), amino acids 30 to 519 of SEQ ID NO: 1 (SEQ ID NO: 41), amino acids 30 to 564 of SEQ ID NO: 1 (SEQ ID NO: 42) and amino acids 31 to 564 of SEQ ID NO: 1 (SEQ ID NO: 43). A protein according to any one of claims 1 to 11, wherein A is an immunogenic fragment of UspA2 having at least 52%, 55%, 57%, 62%, 64%, 69%, 73%, 76%, 81%, 88% or 100% identity with SEQ ID NO: 39. A protein according to any one of claims 1 to 11, wherein A is an immunogenic fragment of UspA2 having at least 52%, 57%, 62%, 65%, 67%, 70%, 73%, 76%, 81%, 100% identity with SEQ ID NO: 43. A protein according to any one of claims 1 to 14, wherein the protein is selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 88. A protein according to any one of claims 1 to 14, wherein A is an immunogenic fragment of UspA2 comprising a laminin binding domain and a fibronectin binding domain. A protein according to any one of claims 1 to 14 wherein A is an immunogenic fragment of UspA2 comprising a laminin binding domain, a fibronectin binding domain and a C3 binding domain. 18. An immunogenic composition comprising a protein of formula (I) as defined in any one of claims 1 to 17. The immunogenic composition of claim 18 comprising an immunogenic fragment of UspA2 selected from the group consisting of amino acids 30 to 540 of SEQ ID NO: 1 (SEQ ID NO: 39), amino acids 31 to 540 of SEQ ID NO: 1 (SEQ ID NO: 40), amino acids 30 to 519 of SEQ ID NO: 1 (SEQ ID NO: 41), amino acids 30 to 564 of SEQ ID NO: 1 (SEQ ID NO: 42) and the amino acids 31 to 564 of SEQ ID NO: 1 (SEQ ID NO: 43). 20. An immunogenic composition according to claim 18 comprising an immunogenic fragment of UspA2 having at least 52%, 55%, 57%, 62%, 64%, 69%, 73%, 76%, 81%, 88% or 100% d identity with SEQ ID NO: 39. 21. An immunogenic composition according to claim 18 comprising an immunogenic fragment of UspA2 having at least 52%, 57%, 62%, 65%, 67%, 70%, 73%, 76%, 81%, 100% identity. with SEQ ID NO: 43. The immunogenic composition of claim 18 comprising a protein selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO. : 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 88. 23. Immunogenic composition according to any one of claims 18 to 22 further comprising at least one Haemophilus influenzae antigen. The immunogenic composition of claim 23, wherein the at least one antigen is D protein. 25. Immunogenic composition according to claims 18 to 24, further comprising the protein E. 26. An immunogenic composition according to any one of claims 18 to 25 further comprising PilA. 27. An immunogenic composition according to claim 26, wherein PE and PilA are present as a fusion protein. 28. A vaccine comprising the protein of any one of claims 1 to 17 or an immunogenic composition according to any one of claims 18 to 27. 29. The vaccine of claim 28 further comprising an adjuvant. 30. The vaccine of claim 29, wherein the adjuvant is AS01E. The vaccine according to any one of claims 28 to 30, wherein the immunogenic composition contains a protein of SEQ ID NO: 69, protein D and a PE-PilA fusion protein. 32. The vaccine according to any one of claims 28 to 31, wherein the PE-PilA fusion protein is LVL-735. A method of treating or preventing otitis media in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an immunogenic composition according to any one of claims 18 to 27 or vaccine according to any one of claims 28 to 32. A method of treating or preventing acute exacerbations of chronic obstructive pulmonary disease (COPD) in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an immunogenic composition according to any one of Claims 18 to 27 or vaccine according to any one of Claims 28 to 32. A method of treating or preventing pneumonia in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an immunogenic composition according to any one of claims 18 to 27 or the vaccine according to any of claims 28 to 32. 36. A method of treating or preventing an infection or illness caused by M. catarrhalis in a subject in need thereof comprising administering to said subject a therapeutically effective amount of an immunogenic composition according to the present invention. any of claims 18 to 27 or the vaccine of any one of claims 28 to 32. 37. Protein according to claims 1 to 17, or immunogenic composition according to claims 18 to 27, or vaccine according to claims 28 to 32, for use in the treatment or prevention of otitis media. 38. A protein according to claims 1 to 17, or an immunogenic composition according to claims 18 to 27, or vaccine according to claims 28 to 32, for use in the treatment or prevention of acute exacerbations of chronic obstructive pulmonary disease (COPD). 39. Protein according to claims 1 to 17, or immunogenic composition according to claims 18 to 27, or vaccine according to claims 28 to 32, for use in the treatment or prevention of pneumonia. 40. Protein according to claims 1 to 17, or immunogenic composition according to claims 18 to 27, or vaccine according to claims 28 to 32, for use in the treatment or prevention of an infection or a disease due to M catarrhalis.